Methods for detecting the differentiation status of cells using 5T4 antigen expression

ABSTRACT

The present invention relates to methods for detecting the differentiation status of stem cells comprising detecting the expression of 5T4 antigen in said stem cells. The present invention also relates to methods for separating populations of undifferentiated or differentiated mammalian stem cells from a mixture of differentiated and undifferentiated stem cells through detection of 5T4 expression.

RELATED APPLICATIONS

This application is a U.S. National Phase Application under 35 U.S.C. §371 of PCT/GB2003/002836, filed Jul. 2, 2003, which claims benefit ofpriority from U.S. patent application Ser. No. 10/485,655, filed Oct. 9,2003, and GB Patent Application 0215287.4, filed Jul. 2, 2002. All threeapplications are hereby incorporated in their entireties as if fully setforth.

FIELD OF THE INVENTION

The present invention relates to the identification that expression of5T4 antigen is switched on during stem cell differentiation.Accordingly, detection of 5T4 expression can be used as a positiveindicator of the differentiation status of stem cells and a negativeindicator of pluripotency.

BACKGROUND TO THE INVENTION

Mammalian stem cells are undifferentiated, primitive cells which havethe ability both to multiply and to differentiate into specific kinds ofcells. Embryos provide a high concentration of stem cells and stem celllines derived from embryos, embryonic stem (ES) cells, are pluripotent,thus possessing the capability of developing into any cell. These cellsare immortal and can be maintained in an undifferentiated state inculture or directed to undergo differentiation into extra embryonic orsomatic lineages. More recently, it has been recognised that embryonicgerm (EG) cells i.e. cells derived from primordial germ cells may havesimilar properties to ES cells. Other stem cells may be derived fromadults and include mesenchymal, epithelial and neural stem cells.

Such stem cells represent a major potential for cell therapies forregenerative medicine as differentiated cells can be generated fortransplantation, may be genetically modified and can be transplanted aspure populations or, following tissue engineering, as tissues orphysiologically functional parts of organs (organoids). ES cells arealso useful models for studying the cellular and molecular biology ofearly development and functional genomics. In vitro culture of stemcells can also provide a useful system for drug screening and drugdiscovery. ES cells derived from mouse embryos are routinely used in anumber of laboratory techniques ranging from gene knockout studies, forexample generating “knock out” mice models, to transplantation therapies(Sato el al. (2001)).

Stem cells are generally difficult to culture in vitro and carefulcontrol of culture conditions, including the appropriate quality ofserum and culture medium, is require. This is particularly important ifsuch cells are to be genetically modified or manipulated to introducegenetic mutations, to be grown on a large scale or to direct theirdifferentation towards specific cell types. In addition, careful controland analysis of the differentiation status is required to ensure thatthe cultured stem cells are suited for their particular use. Theselection of appropriate starting cells for directing appropriatephenotypic differentiation is essential as failure can lead not only toa lack of benefit but also to significant side-effects which can includeproliferation of differentiated cells. In particular, if cells are notfully differentiated at the time of implantation there is always thepossibility of tumour formation. It is therefore clearly important to beable to confirm and select for the undifferentiated integrity ordifferentiation state of cells within a stem cell population.

Some makers of the status of stem cells are known. Markers currentlyused for analysis of the undiffereniated integrity of ES cells includeOct 3/4 (Rathjen et al. (1999)), Rex-1 (Ben-Susbhan et al. (1998)), thecell-surface Forssman antigen (Willison et al. (1978); Ling et al.(1997)) and alkaine phosphatase (Rathjen et al. (1999)) (Table 1). Allthese markers are expressed in undifferentiated ES cells and theirlevels decrease upon differentiation, However, they are not useful forpredicting both the undifferentiated integrity and differentiation stateof ES cells since they decrease relatively slowly following the onset ofdifferentiation (Lake et al. (2000); Rathjen et al. (1999)).Additionally, with the exception of the Forssman antigen, the analysesare destructive to cells and require relatively large numbers of cellsfor RNA extraction.

Removal of leukemia inhibitory factor (LIF) from the medium results inmouse ES cell differentiation (Smith et al. (1992)), characterised bythe upregulation of transcript markers such as fibroblast growthfactor-5 (Fgf-5), zeta globin (ZG) and Flk-1 (Table 3). However, thesemarkers are transiently expressed and present only on a sub-populationof cells thereby limiting their use as single assay markers of ES cellintegrity and differentiation.

To date, there is no marker that can accurately assess both theundifferentiated integrity and differentiated state of stem cells.Current analyses of these parameters are time-consuming, oftendestructive to cells, and require several different markers (Weinhold etal. (2000); Lake et al (2000); Rathjen et al. (1999)). Analysis in asingle, non-destructive assay would be a valuable tool for a wide rangeof ES cell techniques (Lake et al. (2000); Thorey et al. (1998); Niwa etal. (2000); Wakayama et al (1999)).

The 5T4 oncofoetal antigen is a 72 kDa highly glycosylated single passtransmembrane glycoprotein originally isolated from human placentaltrophoblast (Hole, N. & Stern, P. L. (1988); Hole, N. & Stern, P. L.(1990) and Myers, K. A. et al. (1994). 5T4 has been extensivelycharacterised (see, for example, WO 89/07947). It exhibits restrictedexpression patterns in human adult issues, being expressed bytrophoblast and a few specialised adult epithelia, but is upregulated onmany carcinomas, with tumour overexpression correlating with poorerclinical outcome in ovarian, gastric and colorectal cancers. (Southall,P. J. et al. (1990); Wrigley, E. et al. (1995); Starzynska, T. et al.(1994); Starzynska, T. et al. (1998); Mulder, W. M. et al. (1997);Starzynska, T. et al. (1992)). The pattern of 5T4 expression in stemcell populations has not previously been identified.

SUMMARY OF THE INVENTION

The present application identifies that the expression of 5T4-oncofoetalantigen is a positive marker of differentiated ES cells and a negativeindicator of pluripotency. 5T4 protein and mRNA are not detectable inundifferentiated ES cells but are rapidly upregulated in cells derivedfrom all three germ layers following differentiation. Upregulation of5T4 glycoprotein expression correlates with loss of pluripotent markerssuch as OCT-4. Thus, lack of cell-surface 5T4 antigen is a sensitiveindicator of undifferentiated ES cell pluripotency, allowing rapidmonitoring and optimising of ES cell culture conditions. 5T4 antigenexpression on ES cells is unaffected by extended passage, cloning orgrowth on gelatin-treated plates, allowing differentiation analysis fora wide range of ES cell applications. By contrast, ES cell transcriptmarkers Oct-3/4 or Rex-1 (Rathjen et al. (1999); Niwa et al. (2000);Ben-Sushan et al. (1998)) are unable to confirm homogeneous ES cellintegrity since they continue to be expressed in differentiating5T4-positive monolayer cultures. Upregulation of 5T4 antigen insub-optimal culture conditions is also observed.

Accordingly, in one aspect of the invention, there is provided a methodfor detecting the differentiation status of stem cells comprisingdetecting expression of 5T4 antigen wherein lack of expression of 5T4indicates undifferentiated stem cells whereas an increased level ofexpression indicates stem cells which have activated the differentiationpathway. Preferably, said stem cells are mammalian stem cells and, inparticular, ES cells.

“Differentiation status” refers to the stage of differentiation.Initially, stem cells are undifferentiated, pluripotent cells which cangive rise to cells of one or more differentiated cell types. As theyprogress from undifferentiated through to fully differentiated, cellslose their pluripotency and express a more restricted set of genes.Accordingly, the method of the first aspect encompasses a method fordetecting pluripotent stem cells by detecting a lack of expression of5T4 antigen.

Expression of 5T4 antigen can be detected through detection of the 5T4protein or through detection of mRNA transcripts. Techniques fordetecting gene and protein expression are familiar to those skilled inthe art.

“Expression of 5T4” also extends to activation of the 5T4 promoter in aconstruct which can be detected through expression of a reporter gene,as described herein.

As demonstrated herein, the level of 5T4 expression correlates with thedifferentiation status of the stem cells such as ES cells. Thus, anabsence or lack of 5T4 expression is no 5T4 expression or a low ornegligible level of 5T4 expression and indicates that the stem cells areundifferentiated (or pluripotent) whereas an increased amount ofexpression compared to this low level indicates the presence ofdifferentiated cells. Suitably the level of 5T4 expression may bedetermined through comparative studies of stem cells incubated underdifferent conditions. Levels may be expressed as numbers or % ofpositive cells in a stem cell population when measured by FACS-basedtechniques or through quantitative analysis methods such as quantitativeamplification of mRNAs (e.g. RT-PCR) or quantitative determination ofprotein expression (e.g. Western Blotting). Suitable methods aredescribed herein.

Detection of differentiation status can be particularly useful indetermining optimal cell culture conditions for establishing andmaintaining stem cell cultures. For example, and as described herein,cells can be placed in culture conditions and samples of those cellsremoved and tested for 5T4 expression. 5T4 expression is negativelyassociated with optimised undifferentiated culture conditions. Continualmonitoring of 5T4 expression can allow the manipulation of the cultureconditions, using positive and negative 5T4 expression to obtain therequire differentiation status.

Advantageously, detection of 5T4 expression on stem cells is anon-destructive method. The cell surface expression of 5T4 allowsnon-destructive methods for analysis or sorting of viable cellpopulations. This is in contrast to OCT-4 and other transcription factormarkers which require cell permeabilisation for detection and thusdestruction of the cell population.

In a preferred embodiment there is provided a method of detectingdifferentiation status of a population of mammalian stem cellscomprising the steps of:

-   a) taking a sample of cells from said population of mammalian stem    cells;-   b) incubating said sample with an anti-5T4 antibody under conditions    for specific binding of anti-5T4 antibody to 5T4 antigen;-   c) detecting binding of said antibody to said antigen and thereby    detecting presence of 5T4 on cells in the sample wherein presence of    5T4 is indicative of the presence of differentiated cells in the    sample.

Suitably, the method for detecting 5T4 expression is animmunofluorescent technique in which fluorescently labelled anti-5T4antibody is used and detection is through FACS analysis substantially asdescribed herein. In this embodiment, it is preferred that the anti-5T4antibody specifically recognises an extracellularly expressed portion of5T4. The detection of 5T4 antibody or 5T4 tagged antibody by anti-Ig oranti-tag Abs are envisaged.

Suitably, said mammalian stem cells are derived from embryos and includeembryonic stem cells (ES cells), embryonic germ cells or embryonalcarcinoma cells. Other suitable cells are adult stem cells and includemesenchymal, haematopoeitic, neural and epithelial cells. In oneembodiment, said cells are genetically modified stem cells.

Said stem cells are suitably murine, human, primate, porcine, feline orcanine, bovine, ovine although any mammalian stem cells may be used.

In another aspect there is provided use of anti-5T4 antibodies in amethod for detecting differentiation status of mammalian stem cells.

Suitable anti-5T4 antibodies include those known in the art or anyanti-5T4 antibodies that can be raised according to methods known tothose skilled in the art. In one embodiment, the anti-5T4 antibody isthe 9A7 antibody as described herein. In another embodiment, theantibody is an anti-human 5T4 antibody (mAb 5T4) such as that describedin Hole and Stern 1988). Preferably, the anti-5T4 antibody recognisesthe extracellular domain of the 5T4 antigen to facilitate detection of5T4 cell surface expression and thus allow non-destructive detectionmethods (FIG. 4). Methods for labelling antibodies to detect binding areknown to those skilled in the art.

Cultured mammalian stem cells can be used in a number of techniques. Insome techniques it is desirable to use a population of cells comprisingonly differentiated or only undifferentiated cells.

Accordingly, in another aspect of the invention, there is provided amethod for separating a population of undifferentiated or differentiatedmammalian stem cells from a mixture of differentiated andundifferentiated stem cells comprising:

-   a) binding cells in said mixture of differentiated and    undifferentiated stem cells with anti-5T4 antibody,-   b) separating cells with bound antibody from cells with no bound    antibody; and-   c) isolating the cells.

Suitable methods for separating cells include using Ig magnetic beadssuch as MACS beads or other FACS techniques. It will be appreciated thatwhere a population of undifferentiated stem cells is desired, thosecells with no bound antibody may be isolated and selected.

In a preferred embodiment, the cells isolated or separated by saidmethod are viable.

In another embodiment, the antibody is unbound from the cells followingseparation.

As demonstrated herein, with reference to human germ cell tumour cells(embryonal carcinomas), upregulation of 5T4 antigen is observed insub-optimal culture conditions. Accordingly, in a further aspect of theinvention there is provided a method for testing growth serum for itsuse in maintaining mammalian cells comprising detecting expression of5T4.

Suitably, said method comprises the steps of:

-   a) taking mammalian stem cells in culture;-   b) applying test media; and-   c) assessing 5T4 expression in the absence or presence of said media    wherein the presence of 5T4 is an indication that mammalian stem    cells are undergoing differentiation.

This method can be utilized for determining optima cell cultureconditions for establishing and maintaining stem cells in culture.

Stem cells represent useful culture conditions for detecting effects ofa test compound and in particular detecting the ability of a testcompound to induce differentiation or cause any toxic effects.

Accordingly, in a further aspect of the invention, there is provided amethod for detecting the ability of a test compound to induce mammalianstem cell differentiation comprising the steps of:

-   a) incubating a mammalian cell culture in the presence or absence of    said test compound;-   b) detecting 5T4 expression; and-   c) comparing the levels of 5T4 expression in cells wherein increased    5T4 expression in those cells incubated in the presence of said test    compound indicates differentiation induction by said test compound.

In one embodiment, “5T4 expression” may be detected through detecting5T4 promoter activity in a construct in which the 5T4 promoter isoperably linked to a reporter gene as described below. Suitably thereporter gene may be LacZ for detecting in a beta galactosidase system.A number of other suitable reporter gene systems are familiar to thoseskilled in the art.

The detection of 5T4 mRNA and protein expression at the beginning ofstem cell differentiation suggests that activation of 5T4 transcriptionmay be a key event in the induction of differentiation and developmentalpathways.

Thus, the detection of 5T4 expression can be an indication of theinduction of differentiation by a known compound. Suitabledifferentiation-inducing compounds are known to those skilled in theart. Thus, the ability of a test compound to act as an enhancer orinhibitor of the activity of a differentiation-inducing compound can bedetected by measuring 5T4 expression.

Accordingly, in another aspect of the invention, there is provided amethod for detecting the ability of a test compound to enhance orinhibit the activity of a mammalian stem cell differentiation-inducingcompound comprising the steps of:

-   a) incubating a mammalian cell culture treated with a    differentiation-inducing compound in the presence or absence of said    test compound;-   b) detecting 5T4 expression; and-   c) comparing the levels of 5T4 expression in cells wherein increased    5T4 expression in those cells incubated in the presence of said test    compound indicates the ability of a test compound to enhance    differentiation-induction while decreased 5T4 expression indicates    the ability of a test compound to inhibit differentiation-induction.

Transcription of 5T4 may be regulated by interactions at the level ofpromoter activation from the 5T4 gene promoter region. Activation of the5T4 promoter may be harnessed to induce expression of genes at thebeginning of the stem cell differentiation pathway. Suitable genes ofinterest which may be expressed under the control of the 5T4 promoterinclude those which may act as reporter genes, including genes to allowexpression of selectable markers or expression of genes conferringresistance to selectable conditions such as neomycin. Other suitablegenes include functional genes for which expression at the beginning ofdifferentiation may be desirable such as genes involved in specificdifferentiation pathways. In addition, it may be desirable to expressgenes whose products have a toxic effect on a cell. In this way,expression of the gene under control of the 5T4 promoter would induceexpression of a toxic product in those cells undergoing differentiationand therefore eradicate differentiating cells from a population.

Expression from the 5T4 promoter can be induced by introducing a vectorcomprising the 5T4 promoter. Suitably the vector comprising the 5T4promoter can be a targeting construct for homologous recombination.Methods for homologous recombination are well known to those skilled inthe art. Suitable constructs are described, for example, in “GeneTargeting, a practical approach”, Ed. A. L. Joyner, 2^(nd) Edition,Oxford University Press, 2000.

In another aspect of the invention, there is provided a targetingconstruct for homologous recombination targeting the 5T4 promoter.Suitably, the targeting construct comprises a region homologous to 5T4,including immune or human 5T4, flanking a gene of interest. In oneembodiment, the construct further comprises one or more insertionselectors enabling selection of successfully recombined constructs. In aparticularly preferred embodiment, said construct is essentially asshown in FIG. 29.

In another aspect of the invention there is provided a method fordetecting differentiation status of a mammalian stem cell comprising:

-   a) introducing into a stem cell a vector comprising a 5T4 promoter    sequence operably linked to a nucleic acid encoding a reporter gene;    and-   b) detecting an increase in expression of the reporter gene as an    indication of differentiation.

In a further aspect of the invention, there is provided a method ofmodifying a mammalian stem cell comprising introducing a nucleic acidsequence into a mammalian cell such that said nucleic acid sequence isplaced under the control of the 5T4 promoter sequence.

In one embodiment, genes may be expressed under the control of the 5T4promoter region through introduction of vectors comprising the 5T4promoter operably linked to the nucleic acid encoding the gene ofinterest. In another embodiment, the genes introduced may be combined or“knocked in” to the genome of the stem cell through methods such ashomologous recombination. Other suitable methods will be familiar tothose skilled in the art.

In another aspect of the invention, there is provided a method ofmodulating stem cell differentiation comprising modifying the expressionof 5T4 or its functional activity.

Cells which have been sorted according to their expression of 5T4 can beused in a number of stem cell applications. Accordingly, in anotheraspect of the invention, there is provided a use of a stem cell selectedaccording to a method of any of the previous aspects of the invention ina method of treating an individual. Applications of stem cells includetherapeutic applications which are reviewed for example in NatureInsight Review, Vol 414, November 2001. In particular stem cells may betargets for gene therapy and may be genetically modified prior to theiruse in therapeutic applications as described, for example, in Rideout etal. Cell, 109(1):17-27, 2002; Wu et al. Gene Ther 9(4), 245-255,February 2002; Lebkowski et al. Cancer J. 7 Suppl 2; S83-93;November-December 2001.

In another aspect, the methods of the invention may be applicable toconfirming the absence of 5T4-negative i.e. undifferentiated cells froma population prior to introducing said cells into an individual.

In a fiercer aspect of the invention there is provided an isolatedantibody recognising the membrane proximal extracellular domain ofmurine 5T4. Suitably, said antibody is an isolated rat monoclonalanti-5T4 antibody, 9A7 or a human 5T4 specific antibody such as MAb 5T4described by Hole and Stern 1988 (FIG. 4).

Despite human and mouse 5T4 sharing 81% identity in a conserved domainstructure, the monoclonal antibody recognizing human 5T4, sometimesreferred to as MAb5T4, does not cross react with m5T4 (Shaw et al.(2002)). The Mab5T4 antibody recognizes a conformational epitopedependent upon glycosylation and the correct formation of intramoleculardisulphide bonds (Shaw et al. (2002); Hole et al. (1990)).

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and discussion. These aspectsare presented under separate section headings. However, it is to beunderstood that the teachings under each section heading are notnecessarily limited to that particular section heading.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods. See,generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Vol. 194,Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.),McPherson et al., PCR Volume 1, Oxford University Press, (1991), Cultureof Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. L Freshney.1987. Liss, Inc. New York, N.Y.), and Gene Transfer and ExpressionProtocols, pp. 109-128, ed E. J. Murray, The Humana Press Inc., Clifton,N.J.). These documents are incorporated herein by reference.

5T4 antigen is the polypeptide known as 5T4 and characterised, forexample, in WO89/07947. “5T4” may be human 5T4 as characterised by Myerset al ibid., the sequence of which appears in GenBank at accession no.Z29083. A sequence for mouse or murine 5T4 (m5T4) appears in GenBank atAccession no. AJ012160. The organisation of the mouse and human 5T4genes is described, for example, by King et al. Biochim Biophys Acta1999; 1445 (3); 257-70. Canine and feline 5T4 sequences are described,for example, in PCT/GB01/05004 (WO 02/38612)

Sequence analysis of the human 5T4 cDNA identified the antigen as amember of the leucine-rich repeat (LRR) family of proteins (Myers, K. A.et al. (1994)). The protein contains a short cytoplasmic tail of 44amino acids and an extracellular domain consisting of two leucine-richrepeat (LRR) regions with associated cysteine containing flankingregions and separated by a hydrophilic domain. All of the sevenconsensus NxS/T N-glycosylation sites in the extracellular domain areglycosylated with a combination of complex glycans, including two highmannose chains and five sialylated, bi- to tetra-antennary complexchains with minor quantities of core fucosylation (Shaw, D. M. et al.(2002)).

LRR proteins are a diverse family of approximately 60 members, whichhave in common a repeating structure of aXXaXaXXN/C/T, where a is analiphatic residue such as leucine and X is any amino acid (Kobe et al.(1994)). The tertiary structure of porcine ribonuclease inhibitor, whichis comprised entirely of LRRs, has been solved by X-ray crystallography(Kobe et al. (1994)). Ribonuclease inhibitor folds into a horseshoe-likestructure of repeating units of α-helix and β-pleated sheets, thisresolved structure has formed the basis of structural models for otherfamily members (Kajava et al. (1995); Janosi et al. (1999)). However,the precise structure may vary due to differences in the lengths of theLRRs and the presence of other functional domains. Despite no commonfunction having been ascribed, many are involved in protein-proteininteractions and overall it is likely that the LRR domains provide ascaffold for a variety of functions (Kobe et al. (1994), Kobe et al.(1995)).

5T4 antigen is expressed on microvillus projections of cells and whenthe human 5T4 cDNA is constitutively overexpressed in certainfibroblasts or epithelial cells, there are alterations in motility andmorphology which are consistent with a role in both tumour and,trophoblast invasion (Carsberg et al. (1995); Carsberg et al. (1996)).

Sequence comparisons between the human and mouse 5T4 cDNAs (King et al.1999)) indicate the highly conserved structure of 5T4 molecules betweenspecies. These molecules share 81% amino acid identity, with thecytoplasmic and transmembrane domains being completely conserved. Of theseven N-linked glycosylation sites in the human molecule six areconserved in the mouse. The most N-terminal site (N81) is absent, but auadditional site (N334) in the C-terminal flanking region is presentpredicting a similar level of glycosylation to the human molecules. Themurine protein contains an additional six amino acids adjacent to theglycosylation site in the hydrophilic domain, which is a direct repeatof the preceding six amino acids. The expression of 5T4 in trophoblastssuggests it is present at a stage of development common to all mammals.This makes it likely that 5T4 is highly conserved throughout mammals.

As used herein, “differentiated cells” with particular reference to stemcells means cells which retain their characteristic pluripotency ormultipotency i.e. their ability to give rise to all cell types or morethan one differentiated cell type. The terms “differentiated” or“differentiation status” when referring to a cell means cells that havebegun to or have partially or completely developed into cells with adefined phenotype. The characteristic phenotypes of particulardifferentiated cell types are dependent on the particular cell type andare recognised by those skilled in the art.

As used herein, the term “polypeptide” refers to a polymer in which themonomers are amino acids and are joined together through peptide ordisulphide bonds. “Polypeptide” refers to a full-lengthnaturally-occurring amino acid chain or a fragment thereof, such as aselected region of the polypeptide that is of interest in a bindinginteraction, or a synthetic amino acid chain, or a combination thereof.“Fragment thereof” thus refers to an amino acid sequence that is aportion of a full-length polypeptide, between about 8 and about 500amino acids in length, preferably about 8 to about 300, more preferablyabout 8 to about 200 amino acids, and even more preferably about 10 toabout 50 or 100 amino acids in length. Additionally, amino acids otherthan naturally-occurring amino acids, for example β-alanine, phenylglycine and homoarginine, may be included. Commonly-encountered aminoacids which are not gene-encoded may also be used in the presentinvention.

The expression “5T4 antigen” encompasses fragments thereof, andpreferably those fragments having distinct epitopes, and variantsthereof comprising amino acid insertions, deletions or substitutionswhich retain the antigenicity of 5T4. Suitably, the term 5T4 antigen,includes peptides and other fragments of 5T4 which retain at least onecommon antigenic determinant of 5T4.

“Common antigenic determinant” means that the derivative in question hasat least one antigenic fiction of 5T4. Antigenic functions includespossession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against a naturally occurring ordenatured 5T4 polypeptide or fragment thereof, or the ability to bindHLA molecules and induce a 5T4-specific immune response.

Thus 5T4 antigen as referred to herein includes amino acid mutants,glycosylation variants and other covalent derivatives of 5T4 whichretain the physiological and/or physical properties of 5T4. Exemplaryderivatives include molecules wherein the protein of the invention iscovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid. Such a moiety may be a detectable moiety such as an enzyme or aradioisotope. Further included are naturally occurring variants of 5T4found with a particular species, preferably a mammal. Such a variant maybe encoded by a related gene of the same gene family, by an allelicvariant of a particular gene, or represent an alternative splicingvariant of the 5T4 gene.

Derivatives which retain common antigenic determinants can be fragmentsof 5T4. Fragments of 5T4 comprise individual domains thereof, as well assmaller polypeptides derived from the domains. Preferably, smallerpolypeptides derived from 5T4 according to the invention define a singleepitope which is characteristic of 5T4. Fragments may in theory bealmost any size, as long as they retain one characteristic of 5T4.Preferably, fragments will be between 5 and 400 amino acids in length.Longer fragments are regarded as truncations of the full-length 5T4 andgenerally encompassed by the term “5T4”. Advantageously, fragments arerelatively small peptides of the order of 5 to 25 amino acids in length.Preferred are peptides about 9 amino acids in length.

Derivatives of 5T4 also comprise mutants thereof; which may containamino acid deletions, additions or substitutions, subject to therequirement to maintain at least one feature characteristic of 5T4.Thus, conservative amino acid substitutions may be made substantiallywithout altering the nature of 5T4, as may truncations from the 5′ or 3′ends. Deletions and substitutions may moreover be made to the fragmentsof 5T4 comprised by the invention. 5T4 mutants may be produced from aDNA encoding 5T4 which has been subjected to in vitro mutagenesisresulting e.g. in an addition, exchange and/or deletion of one or moreamino acids. For example, substitutional, deletional or insertionalvariants of 5T4 can be prepared by recombinant methods and screened forimmuno-crossreactivity with the native forms of 5T4.

The fragments, mutants and other derivatives of 5T4 preferably retainsubstantial homology with 5T4. As used herein, “homology” means that thetwo entities share sufficient characteristics for the skilled person todetermine that they are similar in origin and function.

“Substantial homology”, where homology indicates sequence identity,means more than 40% sequence identity, preferably more than 45% sequenceidentity and most preferably a sequence identity of 50% or more, asjudged by direct sequence alignment and comparison.

Sequence homology (or identity) may moreover be determined using anysuitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail on theworldwide web at ncbi.nih.gov/BLAST/blasthelp.html, which isincorporated herein by reference.

As used herein, the term “antibody” refers to a polypeptide, at least aportion of which is encoded by at least one immunoglobulin gene, orfragment thereof, and that can bind specifically to a desired targetmolecule. The term includes naturally-occurring forms, as well asfragments and derivatives.

“Specific binding” refers to the ability of two molecular speciesconcurrently present in a heterogeneous (inhomogeneous) sample to bindto one another in preference to binding to other molecular species inthe sample. Typically, a specific binding interaction will discriminateover adventitious binding interactions in the reaction by at leasttwofold, more typically by at least 10-fold, often at least 100-fold;when used to detect analyte, specific binding is sufficientlydiscriminatory when determinative of the presence of the analyte in aheterogeneous (inhomogeneous) sample.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors,plasmids, naked nucleic acids, nucleic acids complexed with polypeptideor other molecules and nucleic acids immobilised onto solid phaseparticles.

A “nucleic acid”, as referred to herein, may be DNA or RNA,naturally-occurring or synthetic, or any combination thereof. Nucleicacids encoding 5T4 antigen may be constructed in such a way that it maybe translated by the machinery of the cells of a host organisms. Thus,natural nucleic acids may be modified, for example to increase thestability thereof. DNA and/or RNA, but especially RNA, may be modifiedin order to improve nuclease resistance. For example, knownmodifications for ribonucleotides include 2′-O-methyl, 2′-fluoro,2′-NH₂, and 2′-O-allyl. Modified nucleic acids may comprise chemicalmodifications which have been made in order to increase the in vivostability of the nucleic acid, enhance or mediate the delivery thereof,or reduce the clearance rate from the body. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions of a given RNA sequence. See, forexample, WO 92/03568; U.S. Pat. No. 5,118,672; Hobbs et al., (1973)Biochemistry 12:5138; Guschlbauer et al., (1977) Nucleic Acids Res.4:1933; Schibaharu et al., (1987) Nucleic Acids Res. 15:4403; Pieken etal., (1991) Science 253:314, each of which is specifically incorporatedherein by reference.

Methods of Detecting 5T4 Expression

The term “expression” refers to the transcription of a gene's DNAtemplate to produce the corresponding mRNA and translation of this mRNAto produce the corresponding gene product (i.e., a peptide, polypeptide,or protein). 5T4 antigen is “expressed” in accordance with the presentinvention by being produced in the cells as a result of translation, andoptionally transcription, of the nucleic acid encoding 5T4. Thus, 5T4 isproduced in situ in the cell. Since 5T4 is a transmembrane protein, theextracellular portion thereof is displayed on the surface of the cell inwhich it is produced.

a) At the RNA Level

Expression levels can be assessed by measuring gene transcription. Thisis preferably carried out by measuring the rate and/or amount ofspecific mRNA production in the cell. RNA may be extracted from cellsusing RNA extraction techniques including, for example, using acidphenol/quanidine isothiocyanate action (RNAzol B; Biogenesis), or RNeasyRNA preparation kits (Qiagen). Typical assay format utilisingribonucleic acid hybridisation include nuclear run-on assays, RT-PCR andRNase protection assays (Melton et al., Nuc. Acids Res. 12:7035).Methods for detection which can be employed include radioactive labels,enzyme labels, chemiluminescent labels, fluorescent labels and othersuitable labels.

Typically, RT-PCR is used to amplify RNA targets. In this process, thereverse transcriptase enzyme is used to convert RNA to complementary DNA(cDNA) which can then be amplified to facilitate detection.

Many DNA amplification methods are known, most of which rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U., et al., Science 242:229-237 (1988) and Lewis, R., GeneticEngineering News 10:1, 54-55 (1990).

PCR is a nucleic acid amplification method described inter alia in U.S.Pat. Nos. 4,683,195 and 4,683,202. PCR can be used to amplify any knownnucleic acid in a diagnostic context (Mok et al., (1994), GynaecologicOncology, 52: 247-252).

A number of alternative amplification technologies including rollingcircle amplification (Lizardi et al., (1998) Nat Genet 19:225) are knownto those skilled in the art.

A primer may be used to allow specific amplification of 5T4 mRNA. Aprobe is e.g. a single-stranded DNA or RNA that has a sequence ofnucleotides that includes between 10 and 50, preferably between 15 and30 and most preferably at least about 20 contiguous bases that are thesame as (or the complement of) an equivalent or greater number ofcontiguous bases of the mRNA of interest.

Primers suitable for use in various amplification techniques can beprepared according to methods known in the art.

Once the nucleic acid has been amplified, a number of techniques areavailable for the quantification of DNA and thus quantification of theRNA transcripts present. Methods for detection which can be employedinclude radioactive labels, enzyme labels, chemiluminescent labels,fluorescent labels and other suitable labels.

Probes may be used to detect the presence of their correspondingsequences through hybridisation reactions e.g. in blotting techniquessuch as northern or southern blotting. The presence of 5T4 nucleic acidsequences may be detected by hybridisation with specific 5T4 probesunder stringent conditions.

The nucleic acid sequences selected as probes should be of sufficientlength and sufficiently unambiguous so that false positive results areminimised. The nucleotide sequences are usually based on conserved orhighly homologous nucleotide sequences or regions of 5T4.

Either the full-length cDNA for 5T4 or fragments thereof can be used asprobes. Preferably, nucleic acid probes are labeled with suitable labelmeans for ready detection upon hybridisation. For example, a suitablelabel means is a radiolabel. The preferred method of labeling a DNAfragment is by incorporating α³²P dATP with the Klenow fragment of DNApolymerase in a random priming reaction, as is well known in the art.Oligonucleotides are usually end-labeled with γ³²P-labelled ATP andpolynucleotide kinase. However, other methods (e.g. non-radioactive) mayalso be used to label the fragment or oligonucleotide, including e.g.enzyme labelling, fluorescent labelling with suitable fluorophores andbiotinylation.

Stringency of hybridisation refers to conditions under which polynucleicacid hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general the stability of a hybrid is a functionof sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na+ at 65-68° C. High stringency conditions can beprovided, for example, by hybridisation in an aqueous solutioncontaining 6×SSC, 5× Denhardt's, 1% SDS (sodium dodecyl sulphate), 0.1Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as nonspecific competitor. Following hybridisation, high stringency washingmay be done in several steps, with a final wash (about 30 min) at thehybridisation temperature in 0.2-0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridisation inthe above-described solution but at about 60-62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove-described solution at about 50-52° C. In that case, the final washis performed at the hybridisation temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds.(1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).Optimal hybridisation conditions have to be determined empirically, asthe length and the GC content of the probe also play a role.

In the context of the present invention, detection of 5T4 expressiongives an indication of differentiation status of mammalian ES cellswhere an increase in 5T4 mRNA expression or stability or both is anindication of induction of differentiation whereas the absence, orexpression at low or negligible levels is an indication ofundifferentiated status.

b) At the Protein Level

Gene expression may also be detected at the protein level by measuringamounts of 5T4 antigen polypeptide. A variety of protocols for detectingand measuring the expression of the amino acid sequences are known inthe art. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, A Laboratory Manual, APS Press, StPaul Minn.) and Maddox D E et al (1983, J Exp Med 15 8:121 1). Asuitable FACS-based method is described in the Examples section herein.

Detection of protein expression may be achieved by using molecules whichbind to the 5T4 antigen polypeptide. Suitable molecules/agents whichbind either directly or indirectly to 5T4 in order to detect thepresence of the protein include naturally occurring molecules such aspeptides and proteins, for example antibodies, or they may be syntheticmolecules.

Other naturally occurring molecules which bind 5T4 include specific 5T4ligands. For example, a number of intracellular partners for 5T4 havebeen identified and are described in Awan et al. (Biochem Biophys ResComm (2002); 290 (3); 1030-1036).

Anti-5T4 antibodies are antibodies that specifically bind to 5T4antigen. They may be polyclonal or monoclonal. If polyclonal antibodiesare desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.)is immunised with an immunogenic polypeptide bearing a 5T4 epitope suchas 5T4-Fc. Serum from the immunised animal is collected and treatedaccording to known procedures. If serum containing polyclonal antibodiesto a 5T4 epitope contains antibodies to other antigens, the polyclonalantibodies can be purified by immunoaffinity chromatography. Techniquesfor producing and processing polyclonal antisera are known in the art.Such antibodies may also be made using polypeptides or fragments thereofhaptenised to another polypeptide for use as immunogens in animals orhumans.

An immune response may also be elicited by immunisation with a vectorcomprising a 5T4-expressing nucleic acid.

The vector employed for immunisation may be any vector, viral ornon-viral. The 5T4 antigen used, whether full length 5T4 or peptidesthereof, may be modified and may be homologous (i.e. derived from thesame species as the subject stem cells) or heterologous in origin.

Monoclonal antibodies directed against 5T4 epitopes can also be readilyproduced by one skilled in the art. The general methodology for makingmonoclonal antibodies by hybridomas is well known. Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. Panels ofmonoclonal antibodies produced against 5T4 epitopes can be screened forvarious properties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phages express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv) and domain antibodies (dAbs) which are described, forexample, in U.S. Pat. No. 6,248,516, U.S. Pat. No. 6,291,158, U.S. Pat.No. 6,172,197 and EP 0,368,684.

Standard laboratory techniques involving antibodies can be used todetect levels of 5T4 in stem cells. One such technique isimmunoblotting, an example of a suitable protocol for which is detailedbelow:

Aliquots of total protein exacts from stem cells (40 μg), are run onSDS-PAGE and electroblotted overnight at 4° C. onto nitrocellulosemembrane. Immunodetection involves antibodies specific for 5T4,appropriate secondary antibodies (goat, anti-rabbit or goat-anti-mouse:Bio-Rad, CA, USA) conjugated to horseradish peroxidase, and the enhancedECL chemilumlinescence detection system (Amersham, UK).

Methods for Selecting Cells by 5T4 Expression

A variety of selection procedures may be applied for the isolation ofcells expressing 5T4 (positive selection) or undifferentiated cellslacking 5T4 expression (negative expression). These include FluorescenceActivated Cell Sorting (FACS), cell separation using magnetic particles,panning, antigen chromatography methods and other cell separationtechniques such as use of polystyrene beads.

Separating cells using magnetic capture may be accomplished byconjugating a molecule which binds to 5T4 antigen to magnetic particlesor beads. For example, the 5T4 binding agent may be conjugated tosuperparamagnetic iron-dextran particles or beads as supplied byMiltenyi Biotec GmbH. These conjugated particles or beads are then mixedwith a cell population which may express 5T4. If a particular cellexpresses 5T4, it will become complexed with the magnetic beads byvirtue of this interaction. A magnetic field is then applied to thesuspension which immobilises the magnetic particles, and retains anycells which are associated with them via the covalently linked antigen.Unbound cells which do not become linked to the beads can be washed awayor collected separately, leaving a population of cells which is isolatedby virtue of the expression of 5T4. Reagents and kits are available fromvarious sources for performing such isolations, and include Dynal Beads(Dynal A S; on the worldwide web at dynal.no), MACS-Magnetic CellSorting (Miltenyi Biotec GmbH; on the worldwide web atmitenylbiotec.com), CliniMACS (AmCell; on the worldwide web atamcell.com) as well as Biomag, Amerlex-M beads and others.

Fluorescence Activated Cell Sorting (FACS) can be used to isolate cellson the basis of their differing surface molecules, for examplesurface-displayed 5T4. Cells in the sample or population to be sortedare stained with specific fluorescent reagents which bind to 5T4. Thesereagents would be the 5T4 binding agent linked (either directly orindirectly) to fluorescent markers such as fluorescein, Texas Red,malachite green, green fluorescent protein (GFP), or any otherfluorophore known to those skilled in the art. The cell population isthen introduced into the vibrating flow chamber of the FACS machine. Thecell stream passing out of the chamber is encased in a sheath of bufferfluid such as PBS (Phosphate Buffered Saline). The stream is illuminatedby laser light and each cell is measured for fluorescence, indicatingbinding of the fluorescent-labelled antigen. The vibration in the cellstream causes it to break up into droplets, which carry a smallelectrical charge. These droplets can be steered by electric deflectionplates under computer control to collect different cell populationsaccording to their affinity for the fluorescent labelled binding agent.In this manner, cell populations which express 5T4 can be easilyseparated from those cells which do not express 5T4. FACS machines andreagents for use in FACS are widely available from sources worldwidesuch as Becton-Dickinson, or from service providers such as ArizonaResearch Laboratories (on the worldwide web at arl.arizona.edu/facs/).

Another method which can be used to separate populations of cellsaccording to cell surface expression of 5T4 is affinity chromatography.In this method, a suitable resin (for example CL-600 Sepharose, PharmacaInc.) is covalently liked to the appropriate 5T4 binding agent. Thisresin is packed into a column, and the mixed population of cells ispassed over the column. After a suitable period of incubation (forexample 20 minutes), unbound cells are washed away using (for example)PBS buffer. This leaves only that subset of cells expressing 5T4 andthese cells are then eluted from the column using (for example) anexcess of the 5T4, or by enzymatically or chemically cleaving the boundreagent from the resin thereby releasing that population of cells whichexhibited 5T4 expression.

Expression from the 5T4 Promoter

The term “promoter” or “promoter region” refers to a nucleic acidsequence, usually found upstream (5′) to a coding sequence, that iscapable of directing transcription of a nucleic acid sequence into mRNA.The promoter or promoter region typically provides a recognition sitefor RNA polymerase and the other factors necessary for proper initiationof transcription. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by inserting or deletingregulatory regions, subjecting the promoter to random or site-directedmutagenesis, etc. The activity or strength of a promoter may be measuredin terms of the amounts of RNA it produces, or the amount of proteinaccumulation in a cell or tissue, relative to a promoter whosetranscriptional activity has been previously assessed.

A “nucleic acid encoding the promoter sequence of 5T4” means a nucleicacid sequence which is capable of directing endogenous transcription of5T4 gene expression. The term moreover includes those polynucleotidescapable of hybridising, under stringent hybridisation conditions, to thenaturally occurring nucleic acids identified above, or the complementthereof.

The phrase “operably linked” refers to the functional spatialarrangement of two or more nucleic acid regions or nucleic acidsequences. For example, a promoter region may be positioned relative toa nucleic acid sequence such that transcription of a nucleic acidsequence is directed by the promoter region. Thus, a promoter region is“operably linked” to the nucleic acid sequence.

A “porter gene” is a gene which is incorporated into an expressionvector and placed under the same controls as a gene of interest toexpress an easily measurable phenotype.

Methods for Detecting Transcription from a Promoter Sequence

Transcription from the 5T4 promoter sequence can be detected using anucleic acid construct comprising the 5T4 promoter sequence operablylinked to a reporter gene. A “reporter gene” is a gene which isincorporated into an expression vector and placed under the samecontrols as a gene of interest to express an easily measurablephenotype. A number of suitable reporter genes are known whoseexpression may be detectable by histochemical staining, liquidscintillation, spectrophotometry or luminometry. Many reporters havebeen adapted for a broad range of assays, including colorimetric,fluorescent, bioluminescent, chemiluminescent, ELISA, and/or in situstaining. Suitable reporter systems are based on the expression ofenzymes such as chloramphenicol acetyltransferase (CAT),beta-galaosidase (beta-gal), beta-glucuronidase, alkaline phosphataseand luciferase. More recently, a number of reporter systems have beendeveloped which are based on using Green fluorescent proteins (GFP) orvarious derivatives or mutant forms including EGFP. Reporter genes anddetection systems are reviewed by Sussman in The Scientist 15 [15]:25,Jul. 23, 2001 which is incorporated by reference.

Vectors for Gene Delivery or Expression.

To generate cells expressing an exogenous gene or 5T4-expressing cells,polypeptides such as 5T4 polypeptides can be delivered by viral ornon-viral techniques. Delivery of 5T4 antigen for immunisation purposescan also be through viral or non-viral techniques.

Non-viral delivery systems include but are not limited to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a 5T4 gene to a target mammalian cell. Thepost-translational modification in relation to phosphorylation orglycosylation may be varied by expression of 5T4 in different targetcells.

Typical transfection methods include electroporation, nucleic acidbiolistics, lipid-mediated transfection, compacted nucleic acid-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), multivalent cations such as spermine, cationic lipids orpolylysine, 1,2,-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 NatureBiotechnology 16: 421) and combinations thereof.

Viral delivery systems include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes viral vectors,retroviral vectors, lentiviral vectors or baculoviral vectors,venezuelan equine encephalitis virus (VEE), poxviruses such as:canarypox virus (Taylor et al 1995 Vaccine 13:539-549), entomopox virus(Li Y et al 1998 XII^(th) International Poxvirus Symposium p144.Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637-46)alphavirus, and alphavirus based DNA vectors.

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

The vector encoding 5T4 may be configured as a split-intron vector. Asplit intron vector is described in PCT patent applications WO 99/15683and WO 99/15684.

If the features of adenoviruses are combined with the genetic stabilityof retroviruses/lentiviruses then essentially the adenovirus can be usedto transduce target cells to become transient retroviral producer cellsthat could stably infect neighbouring cells. Such retroviral producercells engineered to express 5T4 antigen can be implanted in organismssuch as animals or humans for use in the treatment of angiogenesisand/or cancer.

Pox viruses are engineered for recombinant gene expression and for theuse as recombinant live vaccines. This entails the use of recombinanttechniques to introduce nucleic acids encoding foreign antigens into thegenome of the pox virus. If the nucleic acid is integrated at a site inthe viral DNA which is non-essential for the life cycle of the virus, itis possible for the newly produced recombinant pox virus to beinfectious, that is to say to infect foreign cells and thus to expressthe integrated DNA sequence. The recombinant pox virus prepared in thisway can be used as live vaccines for the prophylaxis and/or treatment ofpathologic and infectious disease. Such live vaccines can also be usedto raise antibodies against 5T4. Suitable vectors derived from VacciniaWestern Reserve are described in the Examples section herein.

Expression of 5T4 in recombinant pox viruses, such as vaccinia viruses,requires the ligation of vaccinia promoters to the nucleic acid encoding5T4. Plasmid vectors (also called insertion vectors), have beenconstructed to insert nucleic acids into vaccinia virus throughhomologous recombination between the viral sequences flanking thenucleic acid in a donor plasmid and homologous sequence present in theparental virus (Mackett et al 1982 PNAS 79: 7415-7419). One type ofinsertion vector is composed of: (a) a vaccinia virus promoter includingthe transcriptional initiation site; (b) several unique restrictionendonuclease cloning sites located downstream from the transcriptionalstart site for insertion of nucleic acid; (c) nonessential vacciniavirus sequences (such as the Thymidine Kinase (TX) gene) flanking thepromoter and cloning sites which direct insertion of the nucleic acidinto the homologous nonessential region of the virus genome; and (d) abacterial origin of replication and antibiotic resistance marker forreplication and selection in E. Coli. Examples of such vectors aredescribed by Mackett (Mackett et al 1984, J. Virol. 49: 857-864).

The isolated plasmid containing the nucleic acid to be inserted istransfected into a cell culture, e.g., chick embryo fibroblasts, alongwith the parental virus, e.g., poxvirus. Recombination betweenhomologous pox DNA in the plasmid and the viral genome respectivelyresults in a recombinant poxvirus modified by the presence of thepromoter-gene construct in its genome, at a site which does not affectvirus viability.

As noted above, the nucleic acid is inserted into a region (insertionregion) in the virus which does not affect virus viability of theresultant recombinant virus. Such regions can be readily identified in avirus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton, et al J. Virology 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox andvariola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour virus);avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988) (fowlpox); Boyle,et al Virology 156:355 (1987) (fowlpox); Schnitzlein, et al J.Virological Method, 20:341 (1988) (fowlpox, quailpox); entomopox(Lytvyn, et al J. Gen. Virol 73:3235-3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, HindIII M.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, BamHI J [Jenkins, et al AIDS Research and HumanRetroviruses 7:991-998 (1991)] the EcoRI-HindIII fragment, BamHIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308 220 A1. [Calvert, et alJ. of Virol 67:3069-3076 (1993); Taylor, et al Vaccine 6:497-503 (1988);Spehner, et al (1990) and Boursnell, et al J. of Gen Virol 71:621-628(1990)].

In swinepox preferred insertion sites include the thymidine kinase generegion.

A promoter can readily be selected depending on the host and the targetcell type. For example in poxviruses, pox viral promoters should beused, such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificialconstructs containing appropriate pox sequences can also be used.Enhancer elements can also be used in combination to increase the levelof expression. Furthermore, the use of inducible promoters, which arealso well known in the art, are preferred in some embodiments.

Foreign gene expression can be detected by enzymatic or immunologicalassays (for example, immuno-precipitation, radioimmunoassay, orimmunoblotting). Naturally occurring membrane glycoproteins producedfrom recombinant vaccinia infected cells are glycosylated and may betransported to the cell surface. High expressing levels can be obtainedby using strong promoters.

Stem Cells

Stem cells are undifferentiated, pluripotent, primitive cells with theability both to multiply and differentiate into specific kinds of cells.Mammalian stem cells can also be essentially totipotent (e.g makechimeric mice) as with cell lines derived from mammalian embryos, suchas ES, EG or EC cells, or can be multipotent, typically derived fromadults. Adult-derived stem cells include neural stem cells, mesenchymalstem cells, hematopoeitic stem cells and epithelial stem cells. Stemcell cultures may be genetically modified after isolation and prior totheir differentiation. They could also be modified before generationthough derivation from a suitably genetically modified animal.

Mammalian stem cells may be derived from any mammalian species and thusmay be murine, human or other primate (e.g. chimpanzee, cynomolgusmonkey, baboon, other Old World monkey), porcine, canine, equine, felineetc.

Embryonic stem (ES) cells are stem cells derived from the pluripotentinner cell mass (ICM)/epiblast cells of the pre-implantation,blastocyst-stage embryo. Outgrowth cultures of blastocysts give rise todifferent types of colonies of cells, some of which have anundifferentiated phenotype. If these undifferentiated cells aresub-cultured onto feeder layers they can be expanded to form establishedES cell lines that seem immortal. These pluripotent stem cells candifferentiate in vitro into a wide variety of cell types representativethe three primary germ layers in the embryo. Methods for deriving EScells are known for example from Evans et al. 1981; Nature; 29; 154-156.

Embryonic germ (EG) cell lines are derived from primordial germ cells.Methods for the isolation and culture of these cells are descend, forexample, by McLaren et al. Reprod. Fertil. Dev 2001; 13 (7-8):661-4.Other types of these cells include embryonal carcinoma cells (EC) (asreviewed, for example, in Donovan and Gearhart, Nature 2001; 414(6859):92-97).

Other types of stem cells include cells having haploid genomes asdescribed, for example, in WO 01/32015.

Methods for isolating human pluripotent stem cells are described, forexample, by Trounson, A. O. Reprod. Fertil. Dev 2001; 13 (7-8): 523-32.For example, isolation can require feeder cells (and 20% fetal calfserum) or conditioned medium from feeder cells or feeder cells andmechanical disaggregation (Reubinoff et al 2000). Further methods forproducing pluripotent cells are known from WO 01/30978 where thederivation of pluripotent cells from oocytes containing DNA of all maleor female origin is described. In addition, stem cell-like lines may beproduced by cross species nuclear transplantation as described, forexample, in WO 01/19777, by cytoplasmic transfer to de-differentiaterecipient cells as described, for example, in WO 01/00650 or by“reprogramming” cells for enhanced differentiation capacity usingpluripotent stem cells (see WO 02/14469).

Stem Cell Culture

Cell culture conditions may be modified to favour maintenance of thecells in an undifferentiated state. If conditions are not carefullyselected, stem cells may follow their natural capacity to differentiateinto other cells. ES cells, for example, may differentiate into cellsresembling those of extraembryonic lineages. Few of the factors thatregulate self-renewal of pluripotent stem cells are currently known.Typically, embryonic pluripotent stem cell lines are isolated andmaintained on mitotically inactive feeder layers of fibroblasts or withspecific conditioned medium or, for murine (but not human) ES lines,with leukemia inhibitory factor

Typically, culture systems for ES cells comprise the use of media suchas Dulbecco's modified Eagle's medium (DMEM) as a basal media with theaddition of amino acids and beta mercaptoethanol, serum supplementation(normally Fetal Calf Serum (FCS)), and a embryonic mesenchymal feedercell support layer. Basal media and serum supplements can be obtainedfrom a number of commercial sources. However, any media or serum issubject to variability and even small variations can effect the ES cellculture conditions.

Cells maintained in their undifferentiated state may be subjected tocontrol differentiating conditions to generate cells of the desiredsomatic lineage. Cultured stem cells can be induced to differentiate byseparation of stem cells from feeder cells or by growth of stem cellcolonies in suspension culture to form embryoid bodies which upondissociation can be plated to yield differentiating cells. Conditionsfor obtaining differentiated cultures of somatic cells from ES cells aredescribed, for example, in PCT/AU99/00990. Leukaemia inhibitory factor(LIF) has been identified as one of the factors that can maintainpluripotent stem cells; LIF can replace the requirement for feeder cellsfor murine ES cells (see Nichols et al; (1990) Development 110;1341-1348). Differentiation by removal of LIF is described herein.

For human ES cell lines, growth on primary embryo fibroblasts (pefs) canlimit differentiation. Differentiation can be induced by growth withoutfeeders (pefs) on gelatin-or fibronectin-treated plates. Suitableconditions for differentiation of human ES cells are described herein.

Modulating 5T4 Expression or Activity

The “functional activity” of a protein in the context of the presentinvention describes the function the protein performs in its nativeenvironment. Altering or modulating the functional activity of a proteinincludes within its scope increasing, decreasing or otherwise alteringthe native activity of the protein itself. In addition, it also includeswithin its scope increasing or decreasing the level of expression and/oraltering the intracellular distribution of the nucleic acid encoding theprotein, and/or altering the intracellular distribution of the proteinitself.

The functional activity of 5T4 may be modified by suitablemolecules/agents which bind either directly or indirectly to 5T4, or tothe nucleic acid encoding it. Agents may be naturally occurringmolecules such as peptides and proteins, for example antibodies, or theymay be synthetic molecules. Methods of modulating the level ofexpression of 5T4 include, for example, using antisense techniques.Antisense constructs are described in detail in U.S. Pat. No. 6,100,090(Monia et al), and Neckers et al., 1992, Crit Rev Oncog 3(1-2):175-231,the teaching of which documents are specifically incorporated byreference. Other methods of modulating gene expression are known tothose skilled in the art and include dominant negative approaches aswell as introducing peptides or small molecules which inhibit geneexpression or functional activity.

Uses of Stem Cells

A number of applications for stem cells are known. For example, ES cellsmay be used as an in vitro model for differentiation, especially for thestudy of genes which are involved in the regulation of earlydevelopment. ES cells also have potential utility for germlinemanipulation of livestock animals by using ES cells with or without adesired genetic mutation.

The therapeutic uses of mammalian stem cells are reviewed, for example,in Lovell-Badge, Nature Insight Review, November 2001, 88-91. Some typesof human stem cells, such as bone marrow and skin have been used intherapies for leukemia or skin replacement while others are being usedin trials including fetal midbrain cells for Parkinson's disease, andpancreatic duct cells for diabetes.

A number of uses for mouse ES cells have been demonstrated in animalmodels (as reviewed in Donovan and Gearhart, 2001) and includegeneration of cardiomyocytes to form functioning intracardiac grafts,generation of myelin from glial precursors and the introduction of agenetically modified insulin-producing ES cell line to normaliseglycaemia. Initial results from studies using human pluripotent stemcells in animal models suggest that neuronal cells may be useful intreatment of stroke patients whereas there are number of potentialapplications for mesenchymal-derived stem cells including cardiac musclerepair, bone regeneration and joint repair.

The invention is further described, for the purposes of illustrationonly, in the following examples in which reference is made to thefollowing Figures and Tables:

Table 1 shows the results of FACS analysis of 9A7 activity against apanel of murine cell lines. 10⁵ cells of each line were stained with 9A7and analysed by FACS. Results are representative of three individualcultures and staining experiments.

Table 2. Expression of m5T4 antigen mediates a reduced mean cell volume.FACS was used to assess the forward scatter profile of mid-log phasecultures of the cell lines listed. The geometric mean of the forwardscatter was taken as a measure of average cell volume. These results arerepresentative of three separate experiments.

Table 3. Common makers of ES cell differentiation

Common markers used for determination of ES cell integrity anddifferentiation.

Alkaline phosphatase (Rathjen et al., 1999); Forssman antigen (Ling andNeben, 1997); Oct-3/4, octamer binding protein-3/4 (Lake et al., 2000;Rathjen et al., 1999); Rex-1, reduced expression-1 (Ben-Shushan et al.,1998; Lake et al., 2000; Rathjen et al., 1999); SSEA-1, stage-specificembryonic antigen-1 (Ling and Neben, 1997); Fgf-5, fibroblast growthfactor-5 (Lake et al., 2000; Rathjen et al., 1999); ZG-ζ-globin(Bielinska et al., 1996); Bmp-2, bone morphogenic protein-2 (Weinhold etal., 2000); T-Bra, brachyury (Weinhold et al., 2000); Flk-1, vascularendothelial growth factor receptor-2 (VEGFR-2) (Hirashima et al., 1999);K-18, keratin-18 (Weinhold et al., 2000); Bmp-4, bone morphogenicprotein-4 (Weinhold et al., 2000); NF-68, neurofilament-68k(Itskovitz-Eldor et al., 2000); Vim-vimentin (Weinhold et al., 2000);AFP, α-fetoprotein (Weinhold et al., 2000); TTR Transthyretin;meso-mesoderm (Abe K, 1996). ES-embryonic stem cell; Ecto-ectoderm;Endo-endoderm; Meso-mesoderm.

FIG. 1. The Rabbit anti-m5T4 polyclonal antisera is specific for m5T4 byFACS. Panel A shows the effect of a decreasing concentration m5T4-Fcupon the binding of a constant concentration of Rabαm5T4 to B16 F10-m5T4cells. Cells were analysed by FACS and results expressed as a percentageof the maximal geometric mean. Panels B-D; Grey profiles show A9-m5T4transfectants stained with Rabαm5T4 (1:300 B-D). White profiles showA9-m5T4 (B-rabbit pre-immune serum 1:300), A9H12 neomycin control(C-Rabαm5T4 1:300) and A9-h5T4 (D-Rabαm5T4 1:300).

FIG. 2. Specificity of the 9A7 antibody for m5T4 cDNA transfected cellsby FACS. Grey profiles show A9-m5T4 (9A7, A-C). White profiles showA9m5T4 (rat IgG, A), and A9H12 neomycin (9A7, B), A9-h5T4 transfectants(9A7, C). Panel D shows the effect of a decreasing concentration ofhuman or mouse 5T4-Fc upon the ability of a constant concentration of9A7 to stain A9m5T4 cells. Cells were analysed by FACS and resultsexpressed as a percentage of the maximal geometric mean.

FIG. 3. 9A7 is specific for m5T4 by ELISA. The capacity of variousantigens to inhibit the binding of 9A7 to m5T4-Fc was investigated.Antigen was titrated in a constant concentration of 9A7 (1 μg/ml) andimmediately applied to m5T4-Fc coated plates (1 μg/ml).

FIG. 4. The 9A7 epitope maps to the membrane proximal region of m5T4. A9cell lines expressing human-mouse 5T4 (A and Ci) or mousehuman 5T4chimeric cDNA constructs (B and Cii), in a stable manner, were labelledwith 9A7 (grey profiles) or MAb 5T4 (white profiles). Panel C shows adiagrammatic representation of the 5T4 chimeric molecules. Mousesequences are shown in grey and human sequences in black. From the aminoterminus the domains are labelled; N (amino terminal flanking region),LRR1 (leucine rich region repeat 1), HP (hydrophilic region), LRR2(leucine rich region repeat 2), C (C terminal flanking region), TM(trans-membrane region) and CYT (cytoplasmic domain).

FIG. 5. Biochemical analysis of the 9A7 epitope by Western blot (A) 9A7specificity. Lanes were loaded with 50 ng of human (h) or mouse (m)5T4-Fc fusion protein under reducing (Ai) or non-reducing (Aii)conditions and probed with a rat anti-m5T4 polyclonal antiserum (1:200)or 9A7 (51 g/ml). (B) Carbohydrate and the 9A7 epitope. Lanes wereloaded with 50 ng of m5T4-Fc pre-treated with either nothing (1),sham-treatment (2) or enzyme (3), run under non-reducing conditions andprobed with anti-human IgG-Fc HRP (1:2000) to confirm protein loading or9A7 (51 g/ml) to confirm epitope integrity. (C) Full-length m5T4.Non-reduced Western blot of cell lysates (Ci) and a 9A7immunoprecipitation (Cii) from A9 cells; wild type (wt), neomycincontrol (neo), human (h) or mouse (m) 5T4. Cell lysates were loaded at4×10⁵ cell equivalents/lane (i), and 106 cell equivalents wereimmunoprecipitated with 5 μg of 9A7 with the entire reaction loaded(ii). Both panels (Ci and Cii) were probed with Rabam5T4 (1:3000).

FIG. 6. Distribution of m5T4 at the cell surface. A9h5T4 (A-B), A9m5T4(C-D) and B16 F10-m5T4 (E-F) cells were pre-fixed and stained with MAb5T4 (A-B) or 9A7 (C-F) and analysed by confocal microscopy. Panels show,the entire Z stack projection (A, C, E) or a single Z slice at midpointof Z stack (B, D, F). Each image contains a standard 10 μm bar.

FIG. 7. The distribution of m5T4 after disruption of the cytoskeleton.Cells were left untreated (A) or treated with the cytoskeletal poisonsDemecolcine (B) or Cytochalasin D (C) to disrupt the microtubule networkor the actin fillaments respectively. 2 hours later cells were labelledwith 9A7 and analysed by confocal microscopy. Each image contains astandard 10 μm bar.

FIG. 8. 5T4 antigen expression affects the proliferation and growthpatterns of A9 cells. Panels A, B and C show typical fields of view ofA9H12 Neomycin control cells (A), A9-h5T4 cells (B) and A9-m5T4 cells(C) at 200× magnification. All cultures were seeded in 10% FCS. 24 hrslater the medium was changed to 1% MEM-α and cells cultured for afurther two days before image capture.

FIG. 9. 5T4 expression and cell adhesion. Panel A. 10⁶ cells were seededinto 6-well plates in medium supplemented with 0.25, 1 and 5% FCS. 24hours later the percentage of seeded cells attached was calculated.Panel B. Extracellular matrix proteins and adhesion. 10³ cells wereloaded into protein-coated wells in serum free α-MEM containing 25 μg/mltransferrin. 24 hours later wells were washed and adhesion measured bycrystal violet incorporation.

FIG. 10. The expression of 5T4 cDNA by A9 fibroblasts enhances theirmotility but does not affect their capacity to invade. The relativecapacity of various A9 cell lines to pass across a Matrigel coated(A-invasion) or non-coated tissue culture inserts (B-motility) wasassessed Cells numbers were scored by measurement of incorporatedcrystal violet. Results are expressed as the percentage of all cells,which were present on the lower membrane.

FIG. 11. Immunohistochemical analysis of murine tissues with 9A7.Transverse sections of 17.5 day mouse placenta (A-D) and longitudinalsections of adult mouse brain (E-F) were labelled with rat IgG1 (A, C,E) or 9A7 (B, D, F). Brown colouration represents antibody labelling.Images were captured at 200× magnification.

FIG. 12. Cell surface 5T4 oncofoetal antigen is upregulated on ES cellsfollowing removal of LIF. (a) Cell surface expression of 5T4 on EScells. (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells weredifferentiated for 12 days as monolayer cultures by removal of LIF fromthe growth medium and cell-surface 5T4 measured using rat anti-m5T4monoclonal antibody 9A7 (open population) or control rat IgG (filledpopulation). Primary antibodies were detected using FITC-conjugatedrabbit anti-rat Ig and cell fluorescence measured in a Becton DickinsonFACScan. Viable cells were gated using forward and side scatter and thefigure shows the fluorescence of this population. Percentages indicatethe proportion of the population expressing 5T4. Day 0—undifferentiatedcells; Day 12—12 days following removal of LIF.

(b) Total 5T4 protein expression in ES cell. (i) MESC, (ii) D3, (iii)OKO160 and (iv) 129 ES cells were differentiated for 12 days asmonolayer cultures by removal of LIF from the culture medium, lysed(1.2×10⁷ cells/ml) and 20 μl of the lysate separated by unreducedSDS-PAGE. The membrane was probed using rabbit anti-m5T4 polyclonalserum followed by HRP-conjugated sheep anti-rabbit immunoglobulins anddeveloped by enhanced chemiluminescence. Graphs show the densitometricanalysis of the 5T4 bands, with arbitrary density values on the y-axisand days post-removal of LIF on the x-axis. Controls are mouse A9 cellstransfected with m5T4 cDNA (positive) or vector control (negative).

FIG. 13. Upregulation of 5T4 expression following removal of LIFcorrelates with differentiation of ES cells. (a) Transcript expressionprofiles of ES cells following removal of LIF. (i) MESC, (ii) D3, (iii)OKO160 and (iv) 129 ES cells were differentiated for 12 days asmonolayer cultures by removal of LIF from the growth medium. RNA wasextracted from the cells at the specified time points, DNase treated andcDNA synthesised from the mRNA transcripts. RT-PCR was performed for 35cycles, the samples run on 2% agarose gels containing 400 ng/ml ethidiumbromide and visualised on a L transilluminator. β-tub (housekeepinggene) is included for comparison purposes. To ensure the absence ofgenomic DNA, RT-PCR detection of β-tub was performed on all sampleswithout prior formation of cDNA (mRNA sample). See Table 3 fordescription of markers used. D0—undifferentiated cells; D12—12 daysfollowing removal of LIF. (b) Expression of Forssman antigen on ES cellsfollowing removal of LIF. ES cells were differentiated for 12 days asmonolayer cultures by removal of LIF from the growth medium. Forssmanantigen was determined at the specified time points on differentiating(i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells using ratanti-Forssman antibody (open population) or control rat IgM (closedpopulation), detected as described in the legend to FIG. 12. Viablecells were gated using forward and side scatter and the figure shows thefluorescence of this population. Day 0—undifferentiated cells; Day 12—12days following removal of LIF.

FIG. 14. 5T4 is transcriptionally upregulated on ES cells followingremoval of LIF and the antigen associates with all primary germ layers.(a) 5T4 transcript expression in differentiating ES cells. (i) MESC and(ii) OKO160 ES cell lines were differentiated for 12 days as monolayercultures by removal of LIF from the growth medium. cDNA was prepared atthe specified time points, as described in the legend to FIG. 13, andsemi-quantitative RT-PCR analysis (25 cycles) of m5T4 was performed.Samples were run on 2% agarose gels containing ethidium bromide andvisualised on a UV transilluminator. β-tub (housekeeping gene) isincluded for standardisation. D0—undifferentiated cells; D12—12 daysfollowing removal of LIF.

(b) 5T4 antigen is expressed on cells derived from all three germ layersfollowing removal of LIF from ES cells. MESC ES cells weredifferentiated for 3, 6 and 9 days as monolayer cultures by removal ofLIF from the growth medium and 5T4-positive cells purified usinganti-5T4 monoclonal antibody 9A7 and MidiMACS LS columns. cDNA wasprepared as described above in legend to FIG. 13 followed by RT-PCRanalysis of various germ layer specific transcripts (see Table 3).Samples were run on 2% agarose gels containing ethidium bromide andvisualised on a UV transilluminator. β-tubulin (housekeeping gene) isincluded for standardisation. D3—3 days following removal of LIF; D9—9days following removal of LIF.

FIG. 15. Expression of 5T4 antigen in differentiating ES cells isassociated with the differentiation rate. (a) Expression of 5T4 antigencorrelates with cell migration in differentiating ES cells. (i) MESC and(ii) 129 ES cells (10⁵ cells/3 cm dish) were grown for 0, 3 and 6 daysin DMEMSR in the absence of LIF and viewed under phase contrast on anOlympus inverted microscope. Small arrows show undifferentiated ES cellsand large arrows differentiated/migrating cells. (b) Expression of 5T4correlates with the proliferation rate of differentiating ES cells. (i)MESC and (ii) 129 ES cells (10⁵ cells/3 cm dish) were grown in DMEMSRmedium in the absence (▪) or presence (♦) of LIF (arrow indicates day ofLIF removal) for 3 days and the number of viable cells determined bylight microscopy of cells excluding trypan blue. Bar=10 μM.

FIG. 16. 5T4 antigen is associated with migrating and non-migratingcells in differentiating ES cell cultures. 129 ES cells were grown inmedium containing either differentiation-inducing foetal calf serum(a-c; f-h) or synthetic serum DMEMSR) (d; i-k) in the presence of LIFfor 2 days in gelatin-treated plates. (a-d) 5T4 expression wasdetermined using an Olympus BX-51 fluorescent microscope and imagesoverlaid using Adobe Photoshop. Nuclear staining (DAPI) is shown in blueand 5T4 (FITC) in green. (e) FACS analysis of differentiated (i) andundifferentiated (ii) 129 ES cells demonstrating the presence of cellsurface 5T4 on differentiated cells. (f-k) Confocal images ofdifferentiated (f-h) and undifferentiated (i-k) cells showing 5T4 (f andi; FITC), phase contrast (g and j) and overlay of the 5T4 and phasecontrast images (h and k). Note the lack of nuclei resolution in theundifferentiated phase contrast image (j), probably as a result of thecompacted colony morphology of the undifferentiated ES cells. Bar=10 μM.

FIG. 17. Expression of EGFP-h5T4 in undifferentiated ES cells alterscolony morphology. 129 ES cells were electroporated with 20 μg plasmidDNA and plated into gelatin-treated 9 cm dishes. (a) After 24 h, onethird of the cells were assayed for EGFP expression in a BectonDickinson FACScan (Becton Dickenson; Oxford, UK). Viable cells weregated using forward and side scatter and the figure shows thefluorescence of this population. EGFP positive cells were isolated fromthe remainder of the sample by FACSVantage SE (Becton_Dickenson) andplated out in fresh gelatin-treated 9 cm tissue culture dishes. (b)Cellular localisation of EGFP proteins was determined after 48 h usingan Olympus BX-51 fluorescent microscope. (c) Cell morphology wasdetermined 48 h after transfection using inverted light microscopy.Bar=10 μM.

FIG. 18. Presence of 5T4 on ES cells is a measure of decreasedpluripotency. 129 ES cells were cultured in (a) the presence or (b)absence of LIF for 6 days and (i) SSEA-1 positive cells isolated by FACS(boxed area). (ii) 5T4 expression of the SSEA-1 positive population wasdetermined using antibody 9A7 as described in the legend to FIG. 12 aPluripotency of the SSEA-1 positive cells was determined by chimeraformation following injection of 15 cells into 3.5 day old BL/6blastocysts and subsequent implantation into pseudo-pregnant BDF-1female mice.

FIG. 19 shows 5T4 expression by FACS analysis of human Tera-2 clone 13embryonal carcinoma cells (positive control) and pefs (negativefeeders).

FIG. 20 5T4 oncofoetal antigen expression on GCT27 grown on pef feedersor on gelatin coated dishes.

FIG. 21 5T4 oncofoetal antigen expression on GCT35 grown on pef feedersor on gelatin coated dishes.

FIGS. 22 a and 22 b show morphology of human ES colonies.

FIG. 23 shows FACS analysis of 5T4 expression of cells from“undifferentiated” ES cell colonies and ES cells plated on fibronectincoated dishes.

FIGS. 24 to 27. Illustrate dual 5T4/Oct-4 staining of hES coloniesshowing 5T4 expression is mutually exclusive with OCT-4.

FIG. 28 Confocal microscopy of dual 5T4 and OCT-4 labelling of twodifferentiating ES colonies.

FIG. 29 shows the construct used for homologous recombination in mousestudies. The upper section is a concise restriction map of genomicmurine 5T4; showing the coding sequence of murine 5T4 (shaded region).The lower section is the targeting construct used for homologousrecombination into the 5T4 locus. This shows E. Coli LacZ; PGKDipetheria toxin as negative insertion selector and MCI neo which allowspositive selection of the knock in ES cells. Method as described in GeneTargeting A Practical Approach Ed. AL Joyner, 2^(nd) Edition OxfordUniversity Press 2000.

FIG. 30 β-gal staining of undifferentiated and differentiated 5T4KO/LacZ knock in ES cells

FIG. 31: Expression of cell-surface 5T4 in MESC ES cells differentiatedfor 12 days as suspended embryoid bodies

The invention is further described below, for the purposes ofillustration only, in the following examples.

EXAMPLES Example 1 Generation of m5T4 Specific Antibodies andm5T4-expressing Cell Lines

Materials and Methods

5T4-Fc Fusion Proteins

A 1004 bp cDNA fragment encoding the extracellular domain of mouse 5T4antigen was generated by PCR and cloned by restriction digestion intothe signal-pIg plus expression vector (Ingenious, R&D systems). Stableexpression in Cos-7 cells (Shaw et al. (2000)) was achieved by selectionin G-418 at 1 mg/ml. Mouse and human 5T4-Fc fusion proteins werefractionated from tissue culture supernatant by ammonium sulphateprecipitation and purified by wheatgerm agglutinin and protein Gaffinity chromatography. The concentration was determined by anti-humanFc-capture ELISA (Shaw et al. (2000)) and modified Bradford assayBradford (1976)).

Purity was assessed by silver stained SDS-PAGE. The Fc domain of m5T4-Fcwas removed by overnight digestion with factor Xa protease (Roche). M5T4extracellular domains (m5T4ex) were then enriched by negative selectionon a protein G column and concentrated by centrifugal spin filter (Shawet al. (2002)).

ELISA

Plates were coated with 50 μl of antigen at 1 μg/ml in 0.1M sodiumcarbonate buffer pH 9.3 overnight at 4° C. Plates were washed with PBSTthree times between each layer. Non-specific binding sites were blockedwith 5% milk powder in PBST for 1 hour at 37° C. Plates were incubatedsuccessively for 1 hour at 37° C. with 50 μl per well of each of thefollowing; test sample, biotinylated mouse anti rat κ/λ (1:3000 Sigma)and streptavidin HRP (1:6000 Dako). Reactions were developed with 100 μlof tetra-methyl benzidine at 0.1 mg/ml in 50 mM citrate phosphate bufferpH5.5, stopped by the addition of 50 μl of 1M sulphuric acid and read at450-650 nm.

Polyclonal Antisera

Rabbits were immunised subcutaneously with 100 μg of purified m5T4-Fc inFreunds complete adjuvant and boosted on a fortnightly regime usingFreunds incomplete adjuvant.

Anti-m5T4 activity was assessed by ELISA-based assay against m5T4ex onalternate weeks. Upon acquisition of significant anti-m5T4ex activity,rabbits were terminally bled by cardiac puncture, serum harvested,aliquoted and stored at −20° C.

Cell Culture

Non-adherent cells were grown in RPM 1640 and adherent cells in DMEM(Sigma) supplemented with 2 mM L-glutamine and 10% FCS; transfected celllines were maintained under selection with 1 mg/ml of G-418. Cells weremaintained in a humidified atmosphere of 5% CO₂/air at 37° C. andpassaged on reaching 90% confluence. Four-day conditioned medium wasprepared from confluent cultures of Y3Ag1.2.3. Fusion media comprisedRPMI supplemented to 20% FCS, 50% conditioned medium, 2 mM L-glutamine,2 mM sodium pyruvate and 1×DMEM non-essential amino acids (Sigma).Hybridoma cloning was performed in fusion media supplemented with 10ng/ml human epidermal growth factor.

Flow Cytometry

Adherent cells were removed from flasks with trypsin and washed threetimes at 4° C. with FACS buffer: PBS plus 0.1% BSA and 0.1% sodiumazide. 10⁵ cell aliquots were transferred to a 96 well v-bottom plate,pelleted by centrifugation and the supernatant aspirated. All subsequentsteps were incubated on ice for 30 minutes and cells washed three timeswith FACS buffer between layers. Tissue culture supernatants were testedneat and purified antibodies at 10 μg/ml. Rat and mouse immunoglobulinswere detected with rabbit anti-rat or mouse FITC direct conjugaterespectively (1:30, Dako). Prior to analysis cells were fixed for 10minutes at 4° C. by the addition of an equal volume of 3.7%paraformaldehyde in PBS.

Cell Lines

A9 fibroblastic cells expressing human 5T4 (Carsberg et al. (1995)) orchimeric human-mouse (hm) and mouse-human (mh) 5T4 were generated aspreviously described (Shaw et al. (2002)). Lipofectamine was used totransfect A9 cells with m5T4 cDNA in pCMVα. Bulk cultures were grown fortwo weeks with G-418 at 1 mg/ml and then assessed for 5 m5T4 antigenexpression with the Rabαm5T4 antisera by flow cytometry. Positivecultures were cloned by limiting dilution, assessed for m5T4 antigenexpression as before and positive wells re-cloned. The murine melanomaB16 F10 was transfected by electroporation with human or mouse 5T4 cDNAin pCMVα. Stable expression was achieved by the addition of G-418 at 1mg/ml and clones were established following two rounds of limitingdilution.

Recombinant m5T4 Vaccinia Western Reserve

The full-length m5T4 cDNA (King et al. (1999)) was cloned into theVaccinia transfer plasmid pSC65 (Chakrabati et al. (1997)) such that itis under the control of the synthetic early promoter. Plasmid SC65-m5T4was recombined into the tk locus of the WR strain of vaccinia virususing techniques previously described (Carroll et al. (1998)). Virusstocks were prepared in BSC-1 cells using protocols similar to thatdescribed by Earl et al Earl et al. (1998)).

Immunisation

LOU Rats (Harlan) were immunised twice intra-muscularly with 10⁸ PFUrVV-m5T4 at four-week intervals and test bled two weeks later. Fourweeks after test bleeds were taken, 10⁸ syngeneic splenocytes wereinfected overnight with rVV-m5T4 at a multiplicity of infection of 2 andused to boost the highest responder. On day four post boost this animalwas terminally bled and splenectomised.

Fusion

Cell fusion was performed by the polyethylene glycol method aspreviously described (Kohler et al. (1976)). Fused plasmablasts wereplated at a density of 10⁶/ml in 96 well plates (100 μl per well). After24 hrs in culture 100 μl of fusion medium containing 2×HAT (Sigma) wasadded. The cells were fed at days 4, 7 and 12 by 50% change of 1×HATmedium and on day 14 weaned into HT medium. At day 21, tissue culturesupernatant was removed from wells positive for growth and assayed foranti-m5T4 activity by flow cytometry versus B16 F10-m5T4 or B16 F10-Neocontrol plasmid transfected cells and by ELISA versus m5T4-Fc fusionprotein.

Positive wells were cloned four times by limiting dilution andre-screened as before. Isolated anti-m5T4 antibody isotypes weredetermined with a rat monoclonal antibody isotyping kit according to theinstructions of the manufacturer (The Binding Site).

Antibody Production

Clarified tissue culture supernatant was brought to 45% ammoniumsulphate and stirred overnight at 4° C. The precipitate was pelleted,resuspended in PBS to 10% of the original volume and dialysed at 4° C.against five changes of 100 volumes of PBS. The immunoglobulin waspurified by protein G affinity chromatography and the purified antibodyextensively dialysed against PBS.

Immunoprecipitation, SDS-PAGE and Western Blotting

Cells were lysed at 10⁷ per ml in PBS 0.5% NP40 containing 1× Completeprotease inhibitors (Roche). Lysates were pre-cleared at 4° C. for fourhours with 5 μg of control rat IgG1. Proteins coupled to rat IgG1 werecomplexed with 50 μl of a 50% suspension of Protein G coupled Sepharose(Amersham Biosciences) and removed by centrifugation (1000 g 1 min).

Immunoprecipitations were performed with 5 μg of test antibody, and 50μl of a 50% suspension of protein-G Sepharose. Immunoprecipitates werewashed five times with lysis buffer, resuspended in 50 μl of 1×SDS-PAGEsample buffer and boiled for 3 minutes. Samples were separated bySDS-PAGE using an Atto minigel system according to methods of Laemmli(Laemelli (1970)). Proteins were transferred electrophoretically tonitrocellulose with a Biorad Transblot semidry transfer system andblocked overnight at 4° C. in PBST containing 5% milk powder.

All antibodies were applied for 1 hr at room temperature with agitationand blots washed 5 times for 5 minutes between layers (rat IgG1 and 9A7(10 μg/ml), rabbit anti rat-HRP (1:2000 Dako) andstreptavidin-Horseradish peroxidase (1:6000 Dako). Antibody binding wasdetected by chemiluminesence (Amersham Biosciences) according to theinstructions of the manufacturer.

Immunofluorescence Microscopy

10⁴ Cells were seeded onto acid washed 16 mm glass coverslips in α-MEMcontaining 1% FCS and grown for 48 hrs. Cells were washed three timeswith FACs buffer and fixed with 3.7% paraformaldehyde in PBS for 15 minsprior to labelling or labelled at 4° C. in FACs buffer, washed and thenfixed. Antibodies were applied as follows; 9A7 (10 μg/ml), MAb5T4 (5μg/ml), rat IgG1 (10 μg/ml) or mIgG (5 μg/ml) and the second layerrabbit anti-rat or mouse-FITC conjugate (1:30 Dako as appropriate) for30 mins. Non-fixed samples were then washed and fixed as describedpreviously. Samples were mounted in PBS containing 80% glycerol and 2%1,4-Diazabicyclo[2.2.2]octane, and sealed with clear nail lacquer.

To investigate effect of cytoskeletal disruption upon 5T4 distribution,samples were incubated with 10 μg/ml of either demecolcine orcytochalasin D for two hours prior to labelling (Carsberg et al.(1995)).

Cell Attachment

Aliquots of 3×10⁵ cells were seeded in α.MEM containing 0%, 1%, or 5%FCS in each well of a 6 well plates and incubated for 24 hr. Wells werewashed three times with PBS to remove non-adherent cells and adherentcells trypsinised and counted by haemocytometer.

The effect of extracellular matrix proteins upon cell attachment wasassessed in 96 well plates. Each well was coated with 10 μg of laminin,fibronectin collagen IV or matrigel in PBS overnight at 4° C. Plateswere washed 3 times with PBS and 10³ cells seeded per well in 100 μl ofserum free α.MEM containing 25 μg/ml transferrin (Sigma). Plates wereincubated for 24 hrs, washed 3 times with PBS and stained with 0.01%Crystal Violet in PBS for 15 minutes. Excess dye was removed byextensive washing, plates air dried and residual dye dissolved byagitation for 30 minutes at room temperature with 100 μl per well of 10%acetic acid. The optical density was then read at 570 nm.

Proliferation

Proliferation assays were performed as described (Carsberg et al.(1995); Carsberg et al. (1996)). Briefly, 10⁴ cells were seeded induplicate in 6 well plates in DMEM containing 10% FCS. 24 hours laterthe cells were washed three times and the medium replaced with α-MEMcontaining 0.5%, 1% or 5% FCS. Cells were trypsinised and absolutenumbers determined on at 24-hour intervals with a Coulter counter.

Motility and Invasion Assay

Motility and invasion assays were performed as previously described(Carsberg et al. (1995); Carsberg et al. (1996)). Falcon cell cultureinserts with a non-coated 8 μm porous polyethylene teraphthalatemembrane were used for motility assays, and coated with 10 μg of BiocoatMatrigel for invasion assays (Beckton Dickinson). α-MEM containing 0.25%FCS, used for all assays, was conditioned by incubation with NIH 3T3fibroblasts for 2 hours. 0.5 ml of conditioned medium was placed in thelower compartment and 10⁴ cells seeded in 250 μl of non conditionedmedium in the upper compartment in multiples of four. Twenty-four hourslater wells were washed and fixed with 3.7% paraformaldehyde in PBS for20 minutes.

Migration to the lower chamber was assessed by removal of cells from theupper chamber of membranes (with a cotton bud) and comparison to thetotal number of cells remaining on both surfaces. Cells were stainedwith 0.01% crystal violet and then processed as for cell attachment.

Immunohistochemistry

Murine tissues examined were obtained in triplicate from both male andfemale mice. These included adult heart, lung, liver spleen, kidney,large intestine, small intestine, brain, testes, ovary and 17.5 dayplacenta.

Immunohistochemistry was performed on 5 μm cryostat sections of snapfrozen tissues. Slides were fixed at room temperature for five minutesin acetone and air dried prior to re-hydration in tris buffered saline(TBS: 50 mM tris pH7.6 140 mM NaCl). Endogenous peroxidase activity wasblocked by incubation in TBS containing 0.1% sodium azide and 0.1%hydrogen peroxide, at room temperature for ten minutes. The sectionswere blocked with 10% normal rabbit serum for 30 minutes, all subsequentsteps were in TBS containing 1% normal rabbit serum and incubated for 30minutes at 30° C.

Sections were stained with either 9A7 or a rat IgG1 at 10 μg/ml followedby the secondary antibody, rabbit anti-rat HRP direct conjugate (1:100Dako). Anti-mouse immunoglobulin activity in the secondary antibody wasneutralised by the addition of 10% mouse serum. Immediately prior touse, reagents were spun at 4° C. for 30 minutes at 13,000 rpm in a benchtop microfuge. Antibody labelling was visualised with di-amino benzidineand slides counter-stained, cleared, fixed and mounted as described bySouthall et al (1990).

Polyclonal Rabbit Anti-Mouse 5T4-Fc

To facilitate cloning and preliminary characterisation of m5T4transfected cell lines, a rabbit antiserum was raised against a fusionprotein of the extracellular domain of mouse 5T4 fused to human IgG-Fc(m5T4-Fc). The fourth test bleed from this rabbit showed significantanti-m5T4 activity by ELISA and after boosting, the rabbit wasterminally bled and the serum Harvested. The resulting antiserum(Rabαm5T4) had a titre of 1:5000 by ELISA for the extracellular domainof m5T4 (data not shown).

The rabbit pre-immune serum showed no activity versus control or m5T4transfected cells by flow cytometry (FIG. 1). However, the Rabαm5T4antiserum labelled pCMVα m5T4 cDNA transfected B16 F10-m5T4 melanomacells and A9-m5T4 fibroblasts, but did not label control plasmidtransfected A9H12 cells or h5T4 cDNA transfected A9 fibroblasts FIG. 1).

The binding of Rabαm5T4 to m5T4-Fc or B16 F10-m5T4 cells, as measured byELISA and flow cytometry respectively, was inhibited by pre-incubationwith the m5T4-Fc fusion protein (FIG. 1). This effect was titratable andcould not be replicated with either hIgG or h5T4-Fc (data not shown).These results establish the specificity of Rabαm5T4 antiserum for m5T4by ELISA and flow cytometry (1:300 dilution) and of the expression ofm5T4 molecules on the transfected B16 melanoma and A9 fibroblast celllines.

Although specific at the cell surface, immunohistochemical analysis withRabαm5T4 showed widespread and non-specific staining of mouse placentaland liver sections (data not shown). These reactivities could not beremoved by exhaustive absorption with normal liver tissue and m5T4specific antibodies proved impossible to purify by affinitychromatography. For these reasons monoclonal rat anti-m5T4 antibodieswere generated.

Generation of m5T4 Positive Cell Lines

The establishment of mouse cell lines, which showed stable m5T4expression, was not straightforward. In the A9 cells, flow cytometricanalysis showed stable expression of the m5T4-antigen over 20-25passages. However, after passage 25 the cells began to show evidence ofreduced levels of m5T4 in the population, decreased attachment, reducedproliferation after passage and failure to propagate.

These problems were not encountered during the generation of other A9transfected cell lines expressing human or chimeric 5T4 molecules.Similarly, B16 F10-h5T4 positive cells were relatively easy to produceand maintain whilst B16 F10-m5T4 cell lines required exhaustiveselection to produce cells with stable expression and behaviour invitro. However, as the B16 F10-m5T4 cell line showed uniform growthproperties and stable expression of m5T4 in culture, it was used toscreen hybridoma fusions for rat anti-m5T4 antibodies by flow cytometry.

Monoclonal Antibody Isolation and Characterisation

Rats were immunised with a recombinant strain of Vaccinia West Reserve,which encoded m5T4 (rVV-m5T4) and provided antigen expression in thecontext of a strong adjuvant effect. Two weeks post boost, tail bleedsshowed titres of 1:3000 against m5T4-Fc by ELISA with no crossreactivity towards h5T4-Fc or hIgG (data not shown). Test seraspecifically stained m5T4 transfected cells by flow cytometry and couldonly be blocked from doing so by pre-incubation with m5T4-Fc (data notshown).

The best responder was boosted and the resultant plasmablasts harvestedand fused with the Y3 Ag1.2.3 partner cell line. Of the 960 platedwells, 151 were positive for growth and 104 of these contained ratantibodies, three of which reacted specifically with the m5T4-Fc fusionprotein by ELISA. These wells were designated as 8C7, 9A7 and 10F4 bylocation. However, flow cytometric analysis with the B16 F10-m5T4 cellline, showed that only 9A7 reacted and therefore further analysis waslimited to this antibody.

9A7 activity was specific for A9 cell lines transfected with the m5T4cDNA and did not react with A9 cell lines transfected with eitherneomycin control plasmid (A9H12) or h5T4 cDNA (FIG. 2).

Antibody labelling could be titrated and was inhibited by pre-incubationwith a five fold molar excess of m5T4-Fc (FIG. 2). Similar results wereseen for B16 transfected cells (data not shown). By ELISA, 9A7 onlyrecognised m5T4 as antigen and this recognition could be specificallyinhibited by simultaneous incubation with a five fold molar excess ofm5T4-Fc (FIG. 3). The inhibition of 9A7 binding to m5T4-Fc wastitratable and was not affected by either hIgG or h5T4-Fc. Together,these results confirm the specificity of 9A7 for m5T4 antigen.

Epitope Mapping

Chimeric A9-5T4 cell lines (mh/hm—FIG. 4) were used to map the 9A7epitope to a specific region of the mouse 5T4 molecule. Flow cytometricanalysis showed that the 9A7 and MAb5T4 antibodies labelled the A9-hm5T4and A9-mh5T4 chimeras respectively, in a non-reciprocal fashion (FIG.4). Therefore, both these cell lines expressed antigenically competentchimeric 5T4 molecules. These results localised the MAb5T4 and 9A7epitopes to the membrane proximal regions of the human and mouse 5T4molecule respectively.

Western Blotting and Immunoprecipitation

Reduced and non-reduced Western blots of the mouse and human 5T4-Fcfusion proteins were probed with either 9A7 or a polyclonal ratanti-m5T4 (Ratαm5T4; FIG. 5). Ratαm5T4 reacted specifically with bothreduced and non-reduced m5T4-Fc (FIG. 5A). However, the 9A7 antibody wasonly specific for m5T4-Fc under non-reducing conditions, giving a smallbut significant signal with reduced h5T4-Fc (FIG. 5Aii). Thecontribution of carbohydrate moieties to the integrity of the 9A7epitope was assessed by Western blot analysis of deglycosylated m5T4-Fcmolecules (FIG. 5B). Treatment of m5T4-Fc fusion protein withneuramimidase followed by O-glycosidase produced incremental reductionsin molecular mass, indicating the presence of both sialylation andO-linked glycans. Neither treatment, however, reduced the antigenicityof the 9A7 epitope. However, removal of N-linked carbohydrate moietieswith endoglycosidase H led to a significant reduction in molecular massof the m5T4-Fc molecules with the concomitant ablation of the 9A7epitope (FIG. 5B). The precise glycosylation patterns of the fusionprotein may not reflect the pattern of glycosylation of the nativemolecule, but the antigenicity clearly depends on N-linked sugars. Bycomparison to m5T4-Fc, detection of full-length m5T4 antigen by Westernblotting of m5T4 cDNA-transfected cell lysates with 9A7 is relativelyinsensitive. However, partial purification of membrane glycoproteins bywheatgerm agglutinin enrichment from transfected A9 cell-lysates revealsa broad 72 kDa band specific to the m5T4 cDNA-transfected cells (resultsnot shown). To corroborate this data, non-reduced Western blots of 9A7immunoprecipitates from A9 cell lysates were probed with the Rabαm5T4antiserum. As this antiserum cross-reacts with full-length h5T4 (FIG.5Ci), it can be used to determine the specificity of 9A7immunoprecipitation reactions for human or murine 5T4 molecules. Theresultant 72 kDa band was only present in A9-m5T4 cell lysates,indicating that 9A7 was specific for m5T4 and did not immunoprecipitateh5T4 antigen (FIG. 5Cii).

Cellular Distribution of m5T4

The A9-m5T4 and B16-m5T4 cell lines show a punctate pattern of labellingwhen stained with 9A7 (FIG. 6), which was independent of pre- orpost-fixation and therefore not due to antibody induced antigenredistribution.

Similar patterns of staining were seen by confocal microscopy for themurine mammary carcinoma derived cell lines C127I and EMT6 confirmingthat punctate labelling was independent of CMV immediate early promoterdriven expression.

Disruption of the actin cytoskeleton with cytochalasin D led to aredistribution of punctate staining away from the periphery of the cell.This effect was not seen upon disruption of the microtubule networksuggesting that the integrity of the actin cytoskeleton is an importantfactor in maintaining the distribution of murine 5T4 molecules (FIG. 7).

Cell lines derived from murine tumours were assessed by flow cytometryfor staining with 9A7 (table 1). Positive lines included, three derivedfrom mammary tissue, a squamous lung carcinoma and a teratocarcinomaderived embryonal carcinoma. Those that did not stain with 9A7 includeda fibroblastoid cell line, two melanomas, a lymphoma, two lungarcinomas, a breast carcinoma and also an embryonic stem cell line.

Patterns of Cell Growth

Under low serum conditions A9H12 fibroblasts grow as a “pavement” typemonolayer with many cell-cell contacts with little space between cells(FIG. 8). Transfection of h5T4 into mouse fibroblasts results in a moredendritic morphology, fewer cell-cell contacts and an increased tendencyto disperse (FIG. 8). The expression of m5T4 by A9 fibroblasts resultedin long spindle shaped cells compared to plasmid control transfectedcells (FIG. 8).

M5T4 transfected A9 cells form colonies that stack vertically and alignin a parallel fashion along the axis of the spindle. This results in theformation of “fibres” that grow by extension to connect with others,after which they spread outwards to cover the remaining free surface.This was seen in many experiments, throughout the passage window andwith several independently derived clones.

A9-m5T4 antigen positive cells showed reduced proliferation whencompared to the A9H12 neomycin control cell line. Of the A9-h5T4,A9-m5T4 and A9H12 cell lines, only the A9H12 neomycin cell line could bemaintained in serum free media with doubling time of 75 hours. Additionto the media of FCS (0.5%) allowed all cell lines to be maintained.Proliferation rates were in the order A9H12>A9-h5T4>A9-m5T4 withdoubling time of 62, 120 and 146 hours respectively. Increasing theconcentration of foetal calf serum to 5% did not alter this rank order,but did decrease the differences in doubling times between the lines;A9H12, A9-h5T4 and A9-m5T4 at 53, 62 and 67 hours respectively.

Transfection of the B16 and A9 murine cell lines with m5T4 resulted in a7% reduction of forward scatter as assessed by flow cytometry (Table 2).This implies an average reduction in cell volume upon transfection ofcells with autologous 5T4. This effect was not observed in A9fibroblasts transfected with the h5T4 cDNA, the neomycin controlcassettes or the hm or mh chimeric 5T4 constructs. All cultures showedgood viability with homogeneous 5T4-antigen expression by flowcytometry.

Adhesion

A9 cell lines exhibit serum concentration dependant attachment toplastic (FIG. 9). The degree of this effect lessened as the serumconcentration was increased but the relative differences between celllines remained. The capacity of A9-m5T4 cells to adhere to plastic showsthe most pronounced sensitivity to serum concentration followed byA9-h5T4 and then A9H12.

The extracellular matrix components collagen IV, laminin and fibronectinshowed little differential effect upon adhesion of cells and followedthe same trend as to for adhesion to plastic (FIG. 9). However, matrigelcoated wells resulted in increased adhesion of all cell lines tested butdid not alter their relative propensities to adhere.

Motility and Invasion

The effect of the stable expression of human and mouse 5T4 molecules onthe ability of A9 cells to actively move and invade was compared that ofthe A9H12 neomycin control cell line. The stable expression of human ormouse 5T4 by A9 cells did not significantly alter their propensity toinvade but did increase their motility threefold and sevenfoldrespectively (FIG. 10). These experiments were repeated three timesusing cells of low passage number with uniform growth and 5T4expression. The data presented is representative of these results.Interestingly, cultures of A9-m5T4 positive cells, heterogeneous intheir mouse 5T4 expression and older than 25 passages, show reducedmotility in comparison to homogeneous cultures of lower passage number.

Immunohistochemistry

As the human 5T4-oncofoetal antigen was identified in placental tissue,the immunohistochemical reactivity of anti-m5T4 monoclonal antibodieswas assessed against frozen sections of 17.5-day mouse placenta (FIG.11). This showed that the 9A7 antibody specifically labelled placentaltissue of foetal origin. Cells of the syncitio- and cytotrophoblastshowed discrete staining and the amnion was also positive.

Adult tissues examined were isolated from three individual male andfemale adult mice. These included heart, lung, liver spleen, kidney,large intestine, small intestine, brain, testes and ovary. Limitedstaining of specialised subsets of cells was seen in some of these adulttissues. In order of intensity these were; the choroid plexus in thelateral ventricles of the brain (FIG. 11); the outer epithelial liningof the ovary, the glandular mucosal cells of the large and smallintestine; the glomeruli of the kidney, the sinusoids of the liver, andthe lining of the bronchi.

Adult tissues completely negative for 9A7 staining included the spleen,testis and heart. 9A7 failed to specifically label paraformaldehydefixed wax embedded mouse placenta. 10

Discussion

The production of m5T4 positive cell lines and the description of m5T4expression in the adult mouse required the development of a specificrabbit anti-mouse 5T4-Fc polyclonal serum (Rabαm5T4). Previousobservations had demonstrated the antigenic integrity of the human5T4-Fc fusion protein with both mono and polyclonal reagents (Shaw et al(2000)). Therefore, rabbits were immunised with a m5T4-Fc fusion proteinand the resultant Rabαm5T4 antiserum was shown to be specific for them5T4 antigen at the cell surface in B16 F10 and A9 transfected celllines. However, Rabαm5T4 could not be used for immunohistochemistry dueto high levels of background labelling. Therefore, rats were immunisedwith a vaccinia virus encoding m5T4 antigen and a hybridoma fusionperformed, which was then screened by ELISA and flow cytometry againstthe m5T4-Fc fusion protein and the B16 F10-m5T4 cell line respectively.Screening of this fusion resulted in the isolation of the rat anti-mouse5T4 antibody 9A7. Here we have demonstrated its specificity for the m5T4antigen by flow cytometry, ELISA and immunoprecipitation.

The labelling of tumour and transfected cells lines with 9A7 confirmedexpression of 5T4 antigen by m5T4 mRNA positive cells (King et al.(1999)). The epitope recognised by 9A7, was shown to possess aconformational component and was mapped to the membrane proximal regionof the mouse 5T4 molecule.

Expression of either mouse or human 5T4-cDNA by transfected mouse tumourcell lines increased their motility but reduced their rate ofproliferation and capacity to adhere. The magnitude of these effects wasshown to be serum concentration dependent and was greater when cellswere transfected with autologous 5T4-cDNA.

Finally, the 9A7 antibody was used to describe the distribution of m5T4in adult mouse tissues by immunohistochemistry. Selection for stablegrowth and expression of the m5T4 antigen by murine cell lines wasrelatively difficult. However, the stable expression of human orchimeric 5T4 molecules by these cells was, in comparison, relativelystraightforward yielding stable and long-term expression beyond 25passages. It is possible that over expression of autologous 5T4molecules may deliver negative effects (e.g. through proliferation rateand adhesion changes), which are more pronounced because ofspecies-specific influences of 5T4 antigen expression.

The specificity of 9A7 for m5T4 was confirmed by direct binding andinhibition based assays in vitro (by ELISA) and at the cell surfacewhere binding of 9A7 to m5T4 mRNA positive cells (King et al. (1999))could only be inhibited by the m5T4-Fc fusion protein. Western blots ofm5T4-Fc fusion protein show that reduction significantly lowers itsantigenicity, which implies that the 9A7 epitope, like that of MAb5T4,may be conformational in nature. However, reduced Western blots ofh5T4-Fc revealed a cryptic epitope within the human molecule, which canbe recognised by 9A7.

As the amino acid sequences of human and murine 5T4 show over 81%identity (Myers et al. (1994)) it is likely that the 9A7 epitope, or onevery similar, is present in an altered conformation within h5T4.Reduction, electrophoresis and blotting may allow this cryptic epitopeto refold into a conformation that facilitates recognition by 9A7.

Western blot analysis of full-length m5T4 antigen from cell lysates wasnot very sensitive with 9A7 and required enrichment of membraneglycoproteins by either immunoprecipitation or wheatgerm agglutininaffinity chromatography. Western blots of such enriched cell lysatesshowed a broad 72 kDa band when probed with the Rabαm5T4 antiserum.These results were similar to those previously demonstrated for human5T4 (Hole et al. (1990)) and were limited to m5T4 mRNA positive celllysates (King et al. (1999)). As the Rabαm5T4 antiserum used to probe9A7 immunoprecipitation reactions also detects the human 5T4 antigen byWestern blotting, the lack of a 72 kDa band from h5T4 transfected celllysates indicates that 9A7 specifically immunoprecipitated the m5T4antigen.

The 9A7 epitope was mapped to a region of m5T4 spanning the hydrophilicdomain to the plasma membrane. The MAb5T4 epitope was also shown to mapto this region of human 5T4 and also shows sensitivity to reduction(Shaw et al. (2002), Hole et al. (1990)). Specifically, the 9A7 epitopeis mapped to the LRR2 or the C-terminal flanking region (see e.g. Shawet al. (2002)).

Both m5T4 cDNA transfected and murine tumour derived cell linesexhibited a punctate pattern of labelling with 9A7, which localised tothe cell membrane. This pattern was independent of over-expressiondriven by the CMV immediate early promoter and not induced by antibodymediated re-organisation. However, the disruption of the actincytoskeleton resulted in the redistribution of 9A7 staining, which isconsistent with results reported for human 5T4 antigen (Carsberg et al.(1995)).

Transfection of cells with heterologous 5T4 had a pleiotrophic effect(Carberg et al. (1995); Carsberg et al. (1996)), which was morepronounced upon transfection with autologous 5T4. The morphological,adhesive and proliferative differences between cell lines were clearunder low serum conditions but became less apparent at higher FCSconcentrations. However, under all FCS concentrations examined themorphology, adhesive capacity and proliferation of the A9 cell lines wasalways greatest for A9H12 cells followed by A9-h5T4 and then A9-m5T4.Typically, A9H12 cells show the most adhesive morphology with a“pavement” like appearance and many cell-cell contacts (Carsberg et al.(1996)), whilst A9-m5T4 cells show the least adhesive morphology with aspindle like shape and little contact with the growth support. Both theA9-m5T4 and A9-h5T4 cell lines required >0.1% FCS for growth, whereasA9H12 could be grown short term with no FCS when supplemented withtransferrin. It is likely that the difference in the ability of thesecells to proliferate is linked to their morphology and adhesion to thesubstratum.

The stable expression of human or mouse 5T4 by A9 cells did not altertheir invasive capacity but there is increased motility when compared tocontrol transfected cells. Both the A9 and B16 F10 m5T4 cDNA transfectedcell lines show a reduced mean volume after transfection in comparisonto neomycin control transfected cells. The human ovarian tumour cellline, Hoc-8, also shows a similar reduction in volume whenoverexpressing h5T4 (not shown). As the cytoplasmic and transmembranedomains of the human and mouse 5T4 molecules are completely conserved atthe amino acid level, it is possible that specific interactionsresulting from the extracellular domain of autologous 5T4 molecules maybe involved. Mechanisms reported to affect cell volume include,accelerated cell cycle progression (Lemoine et al, (2001)), modulationof the actin cytoskeleton (Moustakas et al. (1998)) and ion channelmediated regulation of cell hydration (Zhande et al. (1996); Scliess etal. (2000)).

The immunohistochemical distribution of m5T4 antigen in the majority ofmurine adult tissues and 17.5-day placenta, were consistent with thosereported for human 5T4 antigen (Ali et al. (2001); Forsberg et al.(2001)). 9A7 recognised both syncitio- and cytotrophobalst in teemmurine placental tissue, as well as amnion. The 9A7 antibody was alsoshown to label discrete subsets of cells within adult murine tissues.The observation of reactivity in the choroid plexus of the lateralventricals of the brain is novel, as is the above background signalaround the sinusoids of the liver, both of which were not seen in thehuman immunohistochemistry. However, whilst murine brain has been shownto be positive for m5T4 mRNA, no transcripts were detected by Rnaseprotection in murine (King et al. (1999)).

Here we have characterised m5T4 molecules, their tissue expression andtools (antibodies, tumour cells lines) for pre-clinical mouse modelsrelevant to studies of anti-5T4 directed immunotherapy.

Example 2 Expression of 5T4 in Mouse ES Cells

Cell Culture

ES cells were grown in Knockout DE Invitrogen Corporation, Paisley, UK)supplemented with 15% serum replacement (DMEMSR) (D3, MESC and OKO160;Knockout SR, Invitrogen Corporation, Paisley, UK) or 10% foetal calfserum (DMEMFCS) (129; Invitrogen), sodium bicarbonate (0.12% w/v; Sigma,Dorset, UK) L-glutamine (2 mM, Sigma), nucleosides (6 ml of thefollowing solution/500 ml DMEM: adenosine (80 mg), guanosine (85 mg),cytidine (73 mg), uridine (73 mg) and thymidine (24 mg) dissolved in 100ml water, Sigma), 2-mercaptoethanol (50 μM; Life Technologies) and LIF(1000 units/ml of ESGRO; Chemicon Int. Middx. UK) at 37° C./5% CO₂unless otherwise stated (Ward et al. 2002b). 129 (a gift from Dr.Wolfgang Breitwieser, PICR; derived from 129/OLA mice), MESC (a giftfrom Dr. Rhod Elder, PICR; derived from 129/OLA mice) and D3 (AmericanType Culture Collection (ATCC) CRL-1934; derived from 129/Sv+c/+p mice)ES cell lines were grown on irradiated STO fibroblast feeder layers(ATCC). OKO160 ES cell line (a gift from Dr. Austin Smith, Edinburgh,UK) was grown on gelatin-treated plates in the presence of 200 μg/mlG418 due to targeted integration of LacZ in the Oct-4 locus.

All cell lines were plated at approximately 3×10⁶ cells per 10 cm dishand split 1:6 every two days. The media was replenished every day. Forchimera-forming efficiency experiments, 129 ES cells were grown inDMEMSR on gelatin-treated plates in the presence or absence of LIF.Viable cells were determined by exclusion of trypan blue (Sigma, Dorset,UK; 1:4 dilution in PBS).

Differentiation of ES Cells

ES cells were transferred to gelatin-coated plates for 1 day in thepresence of LIF and then replenished with ES media lacking LIF. Themedium was changed daily and monolayer cells passaged before confluency.For determination of cell differentiation rates,), MESC and 129 ES cellswere plated at 10⁵ cells in a 6 well plate in DMEMSR and LIF in theabsence of a feeder layer. There was no significant difference betweenthe plating efficiencies of the two cell lines (data not shown).

Fluorescent Staining of ES Cells

ES cells (5×10⁵ cells/well in a 96-well plate) were incubated with ratanti-mouse 5T4 monoclonal (IgG) antibody 9A7 (Woods A M, 2002), ratmonoclonal (IgM) antibody to MI/22.25 recognising Forssman antigen(Willison and Stern, 1978) or isotype control antibodies (10 μg/ml in0.2% BSA/0.1% sodium azide in PBS) for 1 h on ice. Cells were washed 3times and resuspended in FITC-conjugated rabbit anti-rat Ig for 1 h(1:30 dilution; DAKO, Cambs., UK). Cells were washed twice as descriedabove, fixed in 1% formaldehyde/PBS solution and cell fluorescencemeasured in a Becton Dickinson FACScan.

For fluorescent microscopy, 129 ES cells were cultured in DMEMSR+LIF ongelatin-treated plates and differentiated by addition of mediumcontaining FCS known to result in differentiation of the cells. Thismethod rapidly induces differentiation/cell motility within two days.After 48 h, the media was removed, the cells washed twice in PBS andfixed in paraformaldehyde (4% w/v in PBS) for 15 minutes. Cells wererinsed with PBS and blocking buffer (10% v/v rabbit serum, 0.1% TritonX-100 in TBS) added for 20 min at room temp. Cells were then rinsed inTBS and incubated in rat anti-m5T4 mAb 9A7 or isotype control (10 μg/mlin TBS) for 2 hours at room temp. Cells were rinsed 3 times in TBS, andimmersed in TBS for 20 min at room temp. Cells were then incubated inrabbit anti-rat FITC conjugate (1:30 in TBS; DAKO, UK) for 1 hr at roomtemp. Cells were washed a further 3 times and mounted in VectashieldDAPI mounting medium (Vector Laboratories, Peterborough, UK) and viewedusing an Olympus BX-51 fluorescent microscope or a Zeiss laser scanningconfocal microscope. Images were overlaid using Adobe Photoshop v6.

Expression of EGFP-h5T4 in 129 ES Cells

Cells were grown in DMSR+LIF in the absence of a feeder layer and asub-confluent plate trypsinised, the cells washed in PBS and resuspendedat 1×10⁷ cells/ml in PBS. 20 μg plasmid DNA was added to 0.5 ml of cellsuspension and electroporated at 250V, 475 μF in a BioRad Gene PulserII. After 24 h one third of the cells were assayed for EGFP expressionin a Becton Dickinson FACScan (Becton Dickenson; Oxford, LUK). EGFPpositive cells were isolated from the remainder of the sample byFACSVantage SE (Becton Dickenson) and plated out in fresh gelatintreated 9 cm tissue culture dishes. Cellular localisation of EGFPproteins was determined after 48 h using an Olympus BX-51 fluorescentmicroscope (Olympus, West Midlands, UK). Cell morphology was determined48 h after transfection using inverted light microscopy.

RT-PCR Analysis

RNA was extracted from cells using RNazol B according to themanufacturer's instructions (Biogenesis, Dorset, UK), treated with DNase(Promega, WI, USA) and phenol/chloroform extracted. Synthesis of cDNAfrom mRNA transcripts was performed using the following method: RNA (10μg), dNTP (250 μM), oligo dT (5.0 μg total; Promega, UK), AMV reversetranscriptase (40 units; Promega) in a total volume of 200 μL andincubated at 42° C. for 1 hour. Semi-quantitative RT-PCR of 5T4 wasperformed using 1 μl of the cDNA solution and 25-30 cycles. RT-PCR wasperformed using 5 μl of the cDNA solution and 35 cycles. Samples wererun on 2% agarose gels containing 400 ng/ml ethidium bromide andvisualised on a UV transilluminator. Since the fibroblast feeder layercontains 5T4 transcripts, MESC ES cells were grown for several passageson gelatin-treated plates to remove the fibroblast feeder cells prior tothe extraction of RNA (FIG. 14). Primers used were as follows (read 5′to 3′; forward-F, reverse-R): 5T4 F-aactgccgagtctcagatacc (SEQ ID NO:1),R-atgatacccttccatgtgatcc (SEQ ID NO:2), 55° C. annealing temperature,506 bp; β-tubulin F-tcactgtgcctgaacttacc(SEQ ID NO:3),R-ggaacatagccgtaaactgc (SEQ ID NO:4), 55° C., 317 bp; Fgf-5F-ggcagaagtagcgcgacgtt (SEQ ID NO:5), R-tccggttgctcggactgctt (SEQ IDNO:6), 50° C., 537/515 bp (Johansson and Wiles, 1995); Bmp-2F-gagatgagtgggaaaacg (SEQ ID NO:7), R-gcagtaaaaggcatgatagc (SEQ IDNO:8), 55° C., 606 bp; ζ-globin F-gatgaagaatgagagagc (SEQ ID NO:9),R-agtcaggatagaagacagg (SEQ ID NO:10), 55° C., 406 bp; Oct 3/4F-agaaggagctagaacagtttgc (SEQ ID NO:11), R-cggttacagaaccatactcg (SEQ IDNO: 12), 55° C., 415 bp; Rex-1 F-tgaccctaaagcaagacg (SEQ ID NO:13),R-ataagacaccacagtacacacc (SEQ ID NO:14), 54° C., 414 bp.

Western Blotting

Cells were trypsinised and incubated in tissue culture plates for 30mins at 37° C./5% CO₂ to allow the fibroblast feeder layer to attach tothe plate. The cell suspension was removed, washed in PBS andresuspended in lysis buffer (1×10⁷ cells/ml in 0.5 M Tris, 1.5 M NaCl,0.5% v/v NP-40, 0.2 mM phenylmethylsulfonyl fluoride) on ice for 20min). 20 μl of the lysate was separated by unreduced SDS-PAGE. Positiveand negative controls represent cell lysates of A9 cells transfectedwith either m5T4 cDNA or control vector respectively. Proteins weretransferred onto nitrocellulose membrane using the Novoblot semi-drytransfer system (Amersham Pharmacia, Bucks, UK) and the membrane blockedin 5% milk/0.05% Tween/PBS overnight at 4° C. The membrane was probedusing rabbit anti-m5T4 polyclonal antibody (Woods et al, 2002) followedby HRP-conjugated sheep anti-rabbit immunoglobulins (DAKO, Cambs, UK)and developed by enhanced chemilumenescence (Amersham Pharmacia, UK).Western blot images were captured using an Epi Chemi II Darkroom andSensicam imager with quantification determined by Labworks 4 (UVP, CA,USA).

MACS Separation of 5T4-positive MESC ES Cells

MESC ES cells were grown as described above, trypsinised and washed inPBS. 5T4-positive cells were isolated using mAb 9A7 (10 μg/ml), goatanti-rat Ig magnetic beads and MidiMACS LS columns according to themanufacturer's instructions (Miltenyl Biotech, Surrey, UK).

Determining ES Cell Pluripotency by Chimaeric Mouse Formation

129 ES cells were cultured in DMEMSR on gelatin-treated plates in thepresence or absence of LIF for 6 days and then trypsinized, suspended ingrowth medium at 1×10⁷ cells/ml and incubated with rat anti-mouse SSEA-1(IgM) antibody conjugated with phycoerythrin or an isotype controlantibody (Santa Cruz, Calif.; 1:100 dilution in 0.2% BSA/0.1% sodiumazide in PBS) for 15 minutes on ice. Cells were washed 3 times inculture medium and SSEA-1 positive cells isolated by FACS (FACSVantageSE, Becton Dickenson; Oxford, UK). 5T4 expression of the SSEA-1 positivepopulation was determined as described above. Fifteen SSEA-1 positivecells were injected into each 3.5 day old BL/6 blastocysts and implantedinto pseudo-pregnant BDF-1 female mice (Hogan B, 1994); glasscapillaries from Clark Electromedical Instruments, Kent, UK; Axiovert 10microscope, Carl Zeiss, Herts, UK; MMO-202ND injection manipulation arm,Narishige Int. Ltd., London, UK; Kopf 750 pipette puller, Tunjunga,Calif.). Pluripotency was determined by chimera formation using donorcoat colour. Mice were housed according to Home Office guidelines (1986)and kept on a 12 h-light/dark cycle in which the dark period was from 7pm to 7 am.

Results

The 5T4 Oncofoetal Antigen and mRNA is Upregulated on ES Cells FollowingDifferentiation Induced by Removal of LIF

5T4 antigen is not detected on the surface of undifferentiated ES cellsusing mAb 9A7 (FIG. 12 a). Following withdrawal of LIF for 3 days the5T4 antigen is detected on all the ES cell lines, with the percentage ofpositive cells varying between 7.1% (OKO160) and 50.0% (MESC). Over the12-day differentiation period there is considerable variation in boththe timing of peak 5T4 antigen expression and the proportion of cellslabelling positive between the cell lines. For example, MESC ES cellline exhibits peak expression around day 9 with 85.8% of the populationpositive (FIG. 12 a i), whereas D3 ES cells exhibit a steady increase inpositive cells which peaks at 43.4% on day 12 (FIG. 12 a ii). OKO160 and129 ES cell lines exhibit similar proportions of positive cells at day 3(7.1 and 9.0% respectively) and day 6 (30.6 and 34.0% respectively) andboth cell lines exhibit peak cell staining at day 9 (54.6 and 68.2%respectively). However the proportion of OKO160 cells staining for 5T4antigen is decreased significantly by day 12 (from 54.6% to 17.0%)whereas 129 is only slightly reduced (from 68.2 to 67.3%). Increase intotal 5T4 protein following removal of LIF was confirmed by western blotanalysis of cell lysates using a rabbit anti-m5T4 polyclonal antibody(Woods et al., 2002) (FIG. 12 b). Densitometric analysis of the bandsshows similar expression patterns compared to cell surface 5T4expression, and potential 5T4 isoforms are apparent in the 129 andOKO160 ES cell lines FIG. 12 b iii and iv respectively).

To confirm that upregulation of 5T4 expression upon removal of LIFcorrelates with differentiation of the ES cell lines we assayed variousES cell-specific (Oct 3/4, Rex-1, Forssman antigen) anddifferentiation-specific (Fgf-5, ZG and Bmp-2) markers indifferentiating ES cells FIG. 13). These results show that upregulationof 5T4 correlates with the detection of transcript differentiationmarkers (FIG. 13 a) and a decrease in the ES cell-specific Forssmanantigen (FIG. 13 b), confirming that 5T4 is upregulated during thedifferentiation of ES cells. Most strikingly, the ES cell-associated Oct3/4 and Rex-1 transcripts do not decrease appreciably in MESC, D3 or 129ES cells for at least 12 days following removal of LIF (FIG. 13 a).These transcripts are commonly used to confirm the presence ofundifferentiated ES cells in monolayer culture (Rathjen J, 2002; Rathjenet al., 1999). OKO160 ES cells have a targeted insertion in a singleOct-4 allele, which is likely to account for the relative decrease inOct-3/4 transcripts in this cell line, although Rex-1 transcripts arestill evident 12 days following removal of LIF. There is some disparitybetween the differentiation markers expressed by the ES cell lines (FIG.13 a). For example, Fgf-5 is transiently detected in all but 129 cellsand its peak expression occurs at day 3 in MESC and OKO160 but at day 9in D3 ES cells. Additionally, ZG is transiently detected in all but MESCES cells and peak expression occurs at day 3 in D3 and 129 but at day 9in OKO160 cell lines.

In MESC and D3 cell lines the peak Forssman antigen (FA) expression isobserved in undifferentiated ES cells and decreases upon removal of LIF(FIG. 13 b). However, over the 12-day differentiation period there isconsiderable variation in the expression of the Forssman antigen. Forexample, at 9 days following removal of LIF all ES cell lines exhibit aproportion of cells expressing the antigen. OKO160 and 129 ES cellsexhibit FA staining at day 9 that is only slightly lower than thatpresent on undifferentiated cells. However, at 12 days following removalof LIF the majority of the cell populations are negative for FA,although D3, OKO160 and 129 ES cells exhibit a small proportion ofpositive cells. The differences in FA expression between the cell linesmay be a result of clonal variation or could reflect differentialactivity of specific glycosylating enzymes required for the glycolipidexpression. This data shows that the use of FA as a marker ofundifferentiated ES cells is limited due to the prolonged expression ofthe antigen following removal of LIF in monolayer culture.

The increase in 5T4 antigen on ES cells upon removal of LIF isassociated with increased 5T4 mRNA (FIG. 14 a), probably reflectingtranscriptional upregulation of 5T4. The maximal level of 5T4 transcriptin MESC ES cells (FIG. 14 a i) occurs at day 3, which precedes themaximal level of protein expression (Day 6/9; FIG. 12 a i). The maximalexpression of transcripts in OKO160 cells occurs at day 9 (FIG. 14 aii), which corresponds with maximal protein expression FIG. 12 a iii).There is a clear reduction in transcripts in MESC and OKO160 cell linesafter maximum protein expression. 5T4 transcripts are detected inundifferentiated OKO160 and MESC ES cells by RT-PCR analysis (45 cycles;data not shown), perhaps due to spontaneously differentiating cellswithin the population expressing 5T4 mRNA or low levels of transcriptswithin undifferentiated cells.

ES cells produce differentiated cells corresponding with cell typesrepresentative of the primary germ layers, endoderm, mesoderm andectoderm (Smith, 2001). To determine whether 5T4 is expressed on cellsderived from the three germ layers, MESC ES cells were assayed for thepresence of germ layer-specific transcripts following isolation of the5T4-positive population (FIG. 14 b; Table 3). The detection oftranscripts for AFP, TTR, NF-68, Fgf-5, and T-Bra in the 5T4-positivecell population demonstrates the presence of a proportion of visceralendoderm, endoderm, ectoderm, primitive ectoderm and mesoderm celllineages respectively.

Kinetics of 5T4 Expression Correlates with the Differentiation Rate ofES Cell Lines

With differentiation, DISC show rapid kinetics of 5T4 expressioncompared to the 129 ES cells (FIG. 12 a). This is consistent with therelative proportions of FM positive cells remaining after 12 days ofdifferentiation (FIG. 13 b). When ES cells differentiate there is areduction in the proliferation and increase in apoptosis in thepopulation. As determined by cell numbers in the presence or absence ofLIF, MESC proliferation was clearly reduced after one day followingremoval of LIF whereas 129 ES cells showed no significant change (FIG.15 b i and ii respectively). Thus, the rate of proliferation iscorrelated with the induction of 5T4 expression. In addition, when EScolonies are subject to LIF withdrawal, the outer cells show alteredmorphology and motility (FIG. 15 a). The appearance of such earlydifferentiating cells was more rapid in the MESC than in 129 ES cells.Thus, at three days following removal of LIF, a significant proportionof MESC ES cell colonies exhibited differentiated cells (large arrows)whereas 129 ES cells maintained characteristic ES cell colony morphology(small arrows). By day 6, both ES cell lines exhibited differentiatedcells although the numbers were more numerous in the MESC cell line. Incontrast, to the LIF dependence of MESC, 129 ES cell numbers were notdecreased until 3 days following removal of LIF, suggesting a delayeddifferentiation rate of these cells.

Immunofluorescent analysis of 5T4 expression in undifferentiated anddifferentiated 129 ES cells show that 5T4 is associated with bothcolony-associated and migrating cells (FIG. 16 a-c and f-h). This isconsistent with evidence that expression of 5T4 molecules can influencethe morphology and motility of cells in vitro and suggests a mechanisticinvolvement in the early differentiation process. A significantproportion of the protein appears to be cytoplasmic which may reflectrecent induction of 5T4 protein following differentiation of the cells.Cell-surface 5T4 is clearly present, as demonstrated by the FACS profileof the differentiated cell population FIG. 16 e; 24.0% positive cellscompared to 0.5%).

Expression of EGFP-h5T4 in Undifferentiated ES Cells Alters ColonyMorphology

To further investigate the influence of 5T4 expression on ES cells, 129ES cells were transfected with EGFP, EGFP-h5T4 or EGFP-CD44 plasmids andEGFP-positive cells isolated by FACS (FIG. 17). In the unsortedpopulations, the proportion and intensity of EGFP expression was lowerfor EGFP-h5T4 and EGFP-CD44 compared to EGFP alone (FIG. 17 a). Asexpected, both EGFP-CD44 and EGFP-h5T4 located to the cell membrane andthe majority of EGFP to the nucleus (FIG. 17 b). EGFP-h5T4 transfectedcells also exhibited areas of intense intracellular fluorescence thatare likely to be Golgi-associated (FIG. 17 b). Morphological studiesshowed that ES cells expressing EGFP-h5T4 resulted in increased cellspread compared to the cell surface protein control EGFP-CD44 and EGFPalone (FIG. 17 c). Both EGFP-CD44 and EGFP expressing cells maintainedcharacteristic colony morphologies that were similar to untreated EScells. These results show that expression of 5T4 in differentiatingmouse ES cells is implicated in the spread and movement of the cellsaway from the primary colony.

The differences between the cell-surface EGFP-h5T4 localisation in FIG.17 b and the prominent cytoplasmic 5T4 staining in FIG. 5 a-c is likelyto be due to the use of the CMV promoter in the former. CMV is known tobe highly efficient in undifferentiated ES cells (Ward C M, 2002a),therefore, significant levels of membrane-associated 5T4 would beexpected. In contrast, FIG. 16 demonstrates the localisation of m5T4protein shortly after induction under its natural promoter, and we wouldexpect both cytoplasmic and cell surface 5T4 to be present.

Absence of 5T4 is a Measure of Mouse ES Cell Pluripotency and AllowsOptimisation of ES Cell Growth Conditions

We determined whether 5T4 expression is a useful indicator of lack ofpluripotency in mouse ES cells following removal of LIF compared to theES cell marker SSEA-1 (FIG. 18). Undifferentiated 129 ES cells weresorted for SSEA-1 expression (boxed population in FIG. 18 a i) and werefound to be 5T4 negative (FIG. 18 a ii). The pluripotency of thisSSEA-1+/5T4− population was found to be 52%, as determined by thechimera forming efficiency of the cells following injection into mouseblastocysts and reinplantation into foster mothers (Percentage coatcolour of chimeric mice was 1×60%, 4×25%, 2×20%, 2×10% and 4×<5%).Following removal of LIF from the culture for 6 days, a significantproportion of the cells remained positive for SSEA-1 (FIG. 18 b i) andthese cells were found to be 5T4 positive (FIG. 18 b ii). ThisSSEA-1+/5T4+ cell population exhibited only 7.7% pluripotency (p<0.001compared to SSEA-1+/5T4− population; percentage coat colour of chimericmice was 1×<5%). Furthermore, fewer mice were born in the SSEA-1+/5T4+cell population compared to SSEA-1+/5T4− cells (32.5% and 66%respectively), suggesting differentiated ES cells may be detrimental tothe development process. These results demonstrate that absence of 5T4from an ES cell population is a more accurate and sensitive indicator ofpluripotency than the commonly used ES cell marker SSEA-1.

Many ES cell techniques utilise cloning and expansion of early passagecell lines. Therefore we assayed the effects of cloning and extendedpassage on the expression of the 5T4 antigen to assess its suitabilityas a marker for optimisation of these cells prior to use in suchtechniques. Undifferentiated MESC ES cells did not express cell surface5T4 antigen following culture for 12 passages. Similarly, cloned 129 EScell colonies lacked cell surface antigen following isolation andexpanded growth. Removal of 129 ES cells from a fibroblast feeder layerand subsequent passage on gelatin-treated plates also had no effect on5T4 antigen expression (using DMEMSR+LIF. FIG. 18 a ii). All cloned andextended passage cells exhibited a characteristic increase in cellsurface 5T4 following removal of LIF from the cells, as described inFIG. 12 a.

The quality of serum used for the growth of ES cells is known to affectthe differentiation state of the cells, even in the presence of LIF(Smith, 1992). Growth of 129 ES cells in medium comprising serum inwhich the cells exhibit low cloning efficiency resulted in alteredcolony morphology, increased cell differentiation and induction of 5T4expression compared to cells cultured in normal serum (FIG. 16). We havealso observed some primary embryonic fibroblast (PEF) feeder layerbatches that induce expression of 5T4 on ES cells when co-cultured,suggesting that these PEF batches are not optimal for ES cell growth.The reason for the inability of some PEF batches to sustain ES cells inan undifferentiated state is probably due to harsh passaging (1:10)compared to batches able to maintain undifferentiated cells (passaged1:3). Thus, the absence of 5T4 from ES cells is a useful marker of serumand PEF quality for the undifferentiated growth of these cells.

Discussion

This is the first report of a cell surface marker of ES cellpluripotency that is positively regulated following differentiation ofthe cells. As proof of principle, we show that 5T4 is a more usefulpluripotency marker than SSEA-1 following differentiation of cells byremoval of LIF. This may allow isolation of very early differentiatedcells enabling elucidation of events associated with early ES celldifferentiation. We also demonstrate that kinetics of 5T4 expressioncorrelate with the differentiation rate of ES cells, and we show thatthese rates are varied between ES cell lines. Expression of 5T4 alsocorrelates with the appearance of motile cells, and expression ofEGFP-h5T4 in undifferentiated ES cells leads to increased cell spread.These results suggest that 5T4 is involved in cell motility and/ordecreased cell-cell contacts during the early differentiation of EScells. It further implies an active role for 5T4 during the metastaticprocess and suggests that differentiating mouse ES cells may be usefulfor studying events associated with this process.

Traditionally, markers of ES cell pluripotency are negatively regulated.They are expressed at high levels in undifferentiated ES cells anddecrease following the differentiation (Ben-Shushan et al., 1998; Lingand Neben, 1997; Niwa et al., 2000; Rathjen J, 2002). However, becausethese markers are expressed on a significant proportion of cellsfollowing removal of LIF they are not optimal for accurately determiningpluripotency under these conditions. We have demonstrated that the EScell markers SSEA-1, Oct-4 and Rex-1 (Ben-Shushan et al., 1998; Fan Y,1999; Niwa et al., 2000; Rathjen J, 2002; Rathjen et al., 1999) can bedetected in ES cell populations for at least 12 days following removalof LIF. This is likely to be due to the inefficient differentiation ofES cells in monolayer culture under these conditions. Thus, the kineticsof loss of expression of SSEA-1, Oct-4 or Rex-1 in a differentiating EScell population does not provide for a useful measure of thepluripotency or undifferentiated state of the cells. In contrast, 5T4 ispositively regulated and can rapidly determine the differentiationstate, therefore its absence determines the pluripotency of an ES cellpopulation. Indeed, we have demonstrated that lack of cell surface 5T4on ES cells is a more accurate indicator of pluripotency than SSEA-1,with SSEA1+/5T4+ ES cells showing significantly decreased chimeraforming efficiency.

5T4 antigen is the first cell surface marker that is able to determineboth the pluripotency and early differentiation state of an ES cellpopulation in a single, non-destructive assay. Cell surface 5T4 antigenis also upregulated on cells differentiated as embryoid bodies orfollowing addition of retinoic acid and removal of LIF in monolayercultures (FIG. 31). Thus, 5T4 is a useful marker of differentiation fora range of ES cell techniques. As such, the application of 5T4 as adifferentiation marker of ES cells is most valuable for maintaining anundifferentiated pluripotent population and for establishing optimalgrowth conditions for the cells.

5T4 is unique in that it is both expressed for a relatively prolongedperiod of time and is present on cells derived from each of the threegerm layers. The correlation of 5T4 expression kinetics with thedifferentiation rate of ES cells is an interesting observation that mayenable detailed study of the factors involved in the differentiationprocess. The mechanisms for this correlation are likely to reflectmotility of the differentiated cells away from a primary colony. Theresults demonstrate that there are considerable differences between theES cell lines studied, both in motility and reliance on LIF for cellproliferation.

Furthermore, the transcript expression patterns in differentiating EScells can be different. However, culture conditions or clonal variationwithin populations may account for this difference since the cell linesin this study were not cloned or grown under identical conditions.

5T4 is a member of the LRR family, which contains approximately 60members with no obvious common function (Kobe B, 1994; Kobe B, 1995),and it is likely that the LRR domains of 5T4 provide a scaffold for avariety of biological functions (Shaw D M, 2002). Overexpression of 5T4can have marked effects on both the actin cytoskeleton and motility ofcells (Carsberg et al., 1995; Carsberg et al., 1996; Woods et al.,2002), and it has been shown that the extracellular domain affects cellmotility. The observations that EGFP-h5T4 leads to increasedmotility/spread of ES cells and that 5T4 expression correlates with theappearance of motile cells suggests a motility role for 5T4 during EScell differentiation. Interestingly, the EGFP-h5T4 construct obviatesany role for the terminal cytoplasmic SDV motif of h5T4, which has beenshown to bind through the PDZ domain of TIP-2/GIPC. TIP-2/GIPC is knownto interact with the cytoskeleton through α-actinin which may explainthe cytoskeletal rearrangement phenotype observed when 5T4 is expressed(Awan et al, 2002). There are likely to be additional mechanisms whereby5T4 expression can alter the morphology as well as the motility of cells(Carsberg et al 1995) and these may be of functional significance indevelopment and carcinogenesis.

Example 3

Human Pluripotent Embryonic Cells Show Similar Properties to Murine ESCells for 5T4 Oncofoetal Antigen Expression.

1) Human multipotent GCT (germ cell tumour) 27 and 35 cell lines (Peraet al., Int J Cancer 40: 334-343, 1987; Pera et al., Differentiation 39:139-149, 1988) were grown under conditions which limit thedifferentiation of the embryonal carcinoma stem cell type by growth onprimary embryo fibroblasts. Differentiation was induced by growthwithout feeders on gelatin treated plates.

Methods: Falcon 9 cm tissue culture dishes were coated with 5 ml 0.1%gelatin for 1 h at 37 C. A vial of 129 irradiated (8000 rads) primaryembryo fibroblasts (pees) (4×10⁶) were removed from liquid nitrogen andresuspended in 10 ml of Pef media (Dulbecco's Modified Eagles Medium(DMEM) 2 mM glutamine, 10% FCS). The gelatin was removed, 10 ml of pefsadded to the dish and incubated overnight at 37° C. in humidified 5%CO₂. Before adding GCT cells, the media was changed to Hes media (DMEM,2 mM glutamine, 20% Hyclone defined fetal bovine serum; 90 μM2β-mercaptoethanol; 1% Gibco non-essential amino acid (NEAA) andInsulin, Transferrin and Selinium (ITS) supplement). The medium waschanged daily and cells passaged before confluence. Cells were washedtwice with PBS and then treated with 3 ml trypsin-EDTA (Sigma) for 30seconds, this was removed and the cells incubated for 1 min at 37° C.The cells were harvested in Hes medium and split 1/10 maintained inexponential growth, usually every third day. Differentiation was inducedby plating on to gelatin treated plates and Hes medium with unselectedFCS changed every other day.

5T4 expression was assessed on cell suspensions harvested as above andwashed twice in PBS. Cells at 2×10⁶ cells/ml were added at 100 μl/wellof a 96 well plate and spun at 1000 RPM for 5 mins at 4° C. Thesupernatant was removed, 100 μl monoclonal antibody to human 5T4 antigenat 1 μg/ml or IgG1 isotype control added in FACS buffer (0.2% BSA, 0.1%sodium Azide in PBS) and incubated on ice in dark for 1 h. Followingthree washes in FACS buffer, anti-mouse Ig-FITC second layer antibodywas added and incubated on ice in dark for 45 min. The cells were washedthree times and fixed in 100 μl 4% formaldehyde in PBS before analysison Becton Dickinson FACSCAN.

Results: FIG. 19 shows that mouse primary embryo fibroblasts do notreact with the human 5T4 specific mAb (filled and open areas: control Aband mAb 5T4 respectively). Tera 2 clone 13 cells (Thompson et al., J.Cell Sci. 72: 37-64 1984) are embryonal carcinoma cells with somelimited potency that have been adapted to growth on gelatin coatedtissue culture plates. FACs analysis shows that they are strongly 5T4positive. By contras the embryonal carcinoma stem cells of thepluripotential GCT 27 and GCT 35 lines are 5T4 negative when grown onpefs but rapidly upregulate their 5T4 surface expression when grown ongelatin coated plates (FIGS. 20 and 21).

Conclusion 5T4 surface expression is negatively associated withoptimised undifferentiated culture conditions of germ cell tumourderived embryonal carcinoma cells. In the absence of such conditions thecells will lose their pluripotent phenotype (differentiate) and this isassociated with 5T4 expression.

2) Human embryonic stem (ES) cells (Reubinoff et al, NatureBiotechnology 18:399-404, 2000; ES Cell International) were grown underconditions which limit the differentiation of the ES cell type by growthon primary embryo fibroblasts. Differentiation was induced by growthwithout feeders on fibronectin treated plates. The expression of OCT-4transcription factor was determined as an established marker ofpluripotent embryonic stem cells (Rathjen et al., J. Cell Sci 112:601-12, 1999; Rossant, Stem Cells 19: 477-82, 2001).

Methods: Ten Falcon organ culture plates were coated with 0.1% gelatinfor 1 h at 37° C. Irradiated pefs (1.75×10⁶) were resuspended in 10 mlof pef media and 1 ml added to each plate. The outer reservoir wasfilled with 4 ml of sterile distilled water and the cells incubatedovernight at 37° C. Before adding ES cells, the media was changed to Hesmedia. ES cells were grown and passaged essentially as describedpreviously but without dipase treatment (Reubinoff et al 2000). EScolonies were cut under the microscope using a pulled capillary tube,divided into several pieces and plated on fresh feeder plates. The Hesmedium was changed daily and homogenous ES morphology colonies (FIG. 22a) chosen for passage about every seven days. Some colonies are clearlydistinct with evidence of heterogeneity in morphological types andfurther differentiation is evident if the cells are plated withoutfeeders on fibronectin coated Falcon chamber slide flasks (5 μg/mlovernight at 4° C.) (FIG. 22 b). FACS analyses were performed with cellsuspensions obtained from pooled dissected colonies (20-30) bytrypsin-EDTA treatment (5 min at room temperature followed by gentleagitation after adding Hes medium). In situ expression of 5T4 and OCT 4was performed on fixed cells. Briefly, cells grown on Falcon culturechamber slides were washed with PBS, treated with 4% paraformaldehyde inPBS for 15 mins and washed again with PBS. Non-specific binding wasblocked by incubation with filtered 0.1% BSA, 1% Goat serum, 0.1%Triton-X100 in PBS. Primary antibodies, mouse IgG1 mAb human 5T4 (1μg/ml), mouse IgG2b to human OCT-4 (2 μg/ml; SC-5279, Santa Cruz,Calif.) and isotype control (Biogenesis, UK) were diluted in blockingbuffer and incubated with the cells at room temperature for 2 hours. Theslides were then carefully washed for 5 minutes 4 times in PBS. Secondlayer anti-mouse Ig reagents conjugated with Alexafluor 546 or 488 todetect OCT-4 and 5T4 expression respectively were diluted in blockingbuffer and incubated with the cells for 1 hour at RT. Careful washes inPBS for 2×5 mins, 1×15 mins and 2×5 mins were performed before adding asmall drop of Dapi Vector Shield and cover slipping. The cells wereviewed on an Olympus BX 51 fluorescence microscope and a Zeiss LaserScanning Confocal Microscope. Images were overlaid using Adobe Photoshopversion 6.0.

Results: FIG. 23 a shows that ES cells from colonies harvested fromgrowth on pefs have two distinct populations of 5T4 labelled cells. Thisis consistent with pluripotential ES cells being 5T4 negative and earlydifferentiating populations becoming 5T4 positive. This is supported byabsence of a 5T4 negative population in cells grown under conditionsthat fail to prevent ES differentiation (FIG. 23 b). In situimmunofluorence analysis of both OCT-4 and 5T4 expression in ES coloniesgrown on pefs clearly demonstrates that loss of the pluripotentintracellular OCT-4 expression is congruent with expression of human 5T4(FIG. 24-27). Further analysis of areas of morphologicallydifferentiated cells shows clear evidence of cell surface expression byconfocal microscopy FIG. 28).

Conclusion: Human 5T4 expression is negatively associated withpluripotent human ES cells that express OCT-4. Loss of the latter isaccompanied by up regulation of cell surface 5T4 expression on thedifferentiating cell populations.

Example 4

Additional Murine Studies

We have generated murine ES clones (from E14TG2a ES line) where the LacZgene has been knocked in downstream of the 5T4 promoter. The constructused for homologous recombination is depicted in FIG. 29. The KO 5T4cells show induction of LacZ expression under differentiating cultureconditions detected by using a X-Gal staining kit (Gene Therapy SystemsInc, California (FIG. 30). ES cells on pefs have very few X-Gal stainedcells whereas in differentiating cells grown without feeders and LIFthere are areas of strong staining. Thus reporter genes controlled bythe 5T4 promoter sequences can be used as indicators of desired orundesired ES differentiation.

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All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A method for detecting the differentiation status of cells in apopulation of mammalian pluripotent stem cells comprising detecting cellsurface expression of 5T4 antigen on cells in the population ofmammalian pluripotent stem cells, wherein 5T4 expression indicates cellsin the population that are undergoing differentiation.
 2. A method asclaimed in claim 1 wherein said population of mammalian pluripotent stemcells comprise embryonic stem cells, embryonic germ cells or embryonalcarcinoma cells.
 3. A method as claimed in claim 1 wherein saidmammalian pluripotent stem cells are murine, human, primate, porcine,feline, bovine, ovine or canine.
 4. A method of claim 1 wherein saidcell surface expression of 5T4 is detected by anti-5T4 antibodies.
 5. Amethod of detecting differentiation status of a population of mammalianpluripotent stem cells comprising the steps of: a) obtaining cells fromthe population of mammalian pluripotent stem cells; b) incubating saidcells with a anti-5T4 antibody such that specific binding of anti-5T4antibody to 5T4 antigen occurs; and c) detecting said binding of saidantibody wherein binding of the anti-5T4 antibody to cells is indicativeof the presence of 5T4 and stem cells undergoing differentiation.
 6. Amethod of separating undifferentiated mammalian pluripotent stem cellsfrom mammalian stem cells undergoing differentiation within a populationof mammalian stem cells comprising: a) incubating the population ofmammalian stem cells with anti-5T4 antigen antibody such that theantibody specifically binds to a cell expressing cell surface 5T4antigen; b) separating cells bound to the antibody from cells with nobound antibody; and c) isolating either the bound or unbound cells,wherein cell surface 5T4 antigen expression indicates cells that areundergoing differentiation.
 7. A method as claimed in claim 6 whereinsaid isolated cells are viable.
 8. The method according to claim 5,wherein the anti-5T4 antibody is labeled.
 9. The method according toclaim 6, wherein the anti-5T4 antibody is labeled.
 10. The methodaccording to claim 6, wherein the anti-5T4 antibody is immobilized.